SIMIAN E ADENOVIRUSES SAdV-39, -25.2, -26, -30, -37, AND -38

ABSTRACT

A recombinant vector comprises simian adenovirus SAdV-39, -25.2, -26, -30, -37, and -38 sequences and a heterologous gene under the control of regulatory sequences. A cell line which expresses simian adenovirus SAdV-39, -25.2, -26, -30, -37, and -383 gene(s) is also disclosed. Methods of using the vectors and cell lines are provided.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISC

Applicants hereby incorporate by reference the Sequence Listing materialon the compact discs provided herewith. These compact discs are suppliedin duplicate and contain only the “Sequence Listing” in computerreadable form. These discs are labeled “Copy 1” and “Copy 2,”respectively. The files on these discs are labeled “UPN-U4623 PCTsequence listing.txt”.

BACKGROUND OF THE INVENTION

Adenovirus is a double-stranded DNA virus with a genome size of about 36kilobases (kb), which has been widely used for gene transferapplications due to its ability to achieve highly efficient genetransfer in a variety of target tissues and large transgene capacity.Conventionally, E1 genes of adenovirus are deleted and replaced with atransgene cassette consisting of the promoter of choice, cDNA sequenceof the gene of interest and a poly A signal, resulting in a replicationdefective recombinant virus.

Adenoviruses have a characteristic morphology with an icosahedral capsidconsisting of three major proteins, hexon (II), penton base (III) and aknobbed fibre (IV), along with a number of other minor proteins, VI,VIII, IX, Ma and IVa2 [W. C. Russell, J. Gen Virol., 81:2573-3704(November 2000)]. The virus genome is a linear, double-stranded DNA witha terminal protein attached covalently to the 5′ terminus, which haveinverted terminal repeats (ITRs). The virus DNA is intimately associatedwith the highly basic protein VII and a small peptide pX (formerlytermed mu). Another protein, V, is packaged with this DNA-proteincomplex and provides a structural link to the capsid via protein VI. Thevirus also contains a virus-encoded protease, which is necessary forprocessing of some of the structural proteins to produce matureinfectious virus.

A classification scheme has been developed for the Mastadenovirusfamily, which includes human, simian, bovine, equine, porcine, ovine,canine and opossum adenoviruses. This classification scheme wasdeveloped based on the differing abilities of the adenovirus sequencesin the family to agglutinate red blood cells. The result was sixsubgroups, now referred to as subgroups A, B, C, D, E and F. See, T.Shenk et al., Adenoviridae: The Viruses and their Replication”, Ch. 67,in FIELD'S VIROLOGY, 6^(th) Ed., edited by B. N Fields et al,(Lippincott Raven Publishers, Philadelphia, 1996), p. 111-2112.

Recombinant adenoviruses have been described for delivery ofheterologous molecules to host cells. See, U.S. Pat. No. 6,083,716,which describes the genome of two chimpanzee adenoviruses. Simianadenoviruses, C5, C6 and C7, have been described in U.S. Pat. No.7,247,472 as being useful as vaccine vectors. Other chimpanzeeadenoviruses are described in WO 2005/1071093 as being useful for makingadenovirus vaccine carriers.

What is needed in the art are vectors which effectively delivermolecules to a target and minimize the effect of pre-existing immunityto selected adenovirus serotypes in the population.

SUMMARY OF THE INVENTION

Isolated nucleic acid sequences and amino acid sequences of five novelsimian adenoviruses within subfamily E and vectors containing thesesequences are provided herein. Also provided are a number of methods forusing the vectors and cells of the invention. These adenoviruses includeSAdV-39, SAdV-25.2, SAdV-26, SAdV-30, SAdV-37 and SAdV-38.

The methods described herein involve delivering one or more selectedheterologous gene(s) to a mammalian patient by administering a vector ofthe invention. Use of the compositions described herein for vaccinationpermits presentation of a selected antigen for the elicitation ofprotective immune responses. The vectors based on these simianadenoviruses may also be used for producing heterologous gene productsin vitro. Such gene products are themselves useful for a variety ofpurposes such as are described herein.

These and other embodiments and advantages of the invention aredescribed in more detail below.

DETAILED DESCRIPTION OF THE INVENTION

Novel nucleic acid and amino acid sequences from simian adenovirus 39,SAdV-25.2, SAdV-26, SAdV-30, SAdV-37 and SAdV-38, all of which wereisolated from chimpanzee feces, are provided.

Also provided are novel adenovirus vectors and packaging cell lines toproduce vector based on these sequences for use in the in vitroproduction of recombinant proteins or fragments or other reagents.Further provided are compositions for use in delivering a heterologousmolecule for therapeutic or vaccine purposes. Such therapeutic orvaccine compositions contain the adenoviral vectors carrying an insertedheterologous molecule. In addition, the novel SAdV sequences are usefulin providing the essential helper functions required for production ofrecombinant adeno-associated viral (AAV) vectors. Thus, helperconstructs, methods and cell lines which use these sequences in suchproduction methods, are provided.

The term “substantial homology” or “substantial similarity,” whenreferring to a nucleic acid or fragment thereof, indicates that, whenoptimally aligned with appropriate nucleotide insertions or deletionswith another nucleic acid (or its complementary strand), there isnucleotide sequence identity in at least about 95 to 99% of the alignedsequences.

The term “substantial homology” or “substantial similarity,” whenreferring to amino acids or fragments thereof, indicates that, whenoptimally aligned with appropriate amino acid insertions or deletionswith another amino acid (or its complementary strand), there is aminoacid sequence identity in at least about 95 to 99% of the alignedsequences. Preferably, the homology is over full-length sequence, or aprotein thereof, or a fragment thereof which is at least 8 amino acids,or more desirably, at least 15 amino acids in length. Examples ofsuitable fragments are described herein.

The term “percent sequence identity” or “identical” in the context ofnucleic acid sequences refers to the residues in the two sequences thatare the same when aligned for maximum correspondence. Where gaps arerequired to align one sequence with another, the degree of scoring iscalculated with respect to the longer sequence without penalty for gaps.Sequences that preserve the functionality of the polynucleotide or apolypeptide encoded thereby are more closely identical. The length ofsequence identity comparison may be over the full-length of the genome(e.g., about 36 kbp), the full-length of an open reading frame of agene, protein, subunit, or enzyme [see, e.g., the tables providing theadenoviral coding regions], or a fragment of at least about 500 to 5000nucleotides, is desired. However, identity among smaller fragments, e.g.of at least about nine nucleotides, usually at least about 20 to 24nucleotides, at least about 28 to 32 nucleotides, at least about 36 ormore nucleotides, may also be desired. Similarly, “percent sequenceidentity” may be readily determined for amino acid sequences, over thefull-length of a protein, or a fragment thereof. Suitably, a fragment isat least about 8 amino acids in length, and may be up to about 700 aminoacids. Examples of suitable fragments are described herein.

Identity is readily determined using such algorithms and computerprograms as are defined herein at default settings. Preferably, suchidentity is over the full length of the protein, enzyme, subunit, orover a fragment of at least about 8 amino acids in length. However,identity may be based upon shorter regions, where suited to the use towhich the identical gene product is being put.

As described herein, alignments are performed using any of a variety ofpublicly or commercially available Multiple Sequence Alignment Programs,such as “Clustal W”, accessible through Web Servers on the interne[Thompson et al, 1994, Nucleic Acids Res, 22, 4673-4680]. Alternatively,Vector NTI® utilities [InVitrogen] are also used. There are also anumber of algorithms known in the art that can be used to measurenucleotide sequence identity, including those contained in the programsdescribed above. As another example, polynucleotide sequences can becompared using Fasta, a program in GCG Version 6.1. Fasta providesalignments and percent sequence identity of the regions of the bestoverlap between the query and search sequences. For instance, percentsequence identity between nucleic acid sequences can be determined usingFasta with its default parameters (a word size of 6 and the NOPAM factorfor the scoring matrix) as provided in GCG Version 6.1, hereinincorporated by reference. Similarly programs are available forperforming amino acid alignments. Generally, these programs are used atdefault settings, although one of skill in the art can alter thesesettings as needed. Alternatively, one of skill in the art can utilizeanother algorithm or computer program that provides at least the levelof identity or alignment as that provided by the referenced algorithmsand programs.

“Recombinant”, as applied to a polynucleotide, means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a polynucleotide found in nature. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide. The terms respectively include replicates of theoriginal polynucleotide construct and progeny of the original virusconstruct.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is being compared. Forexample, a polynucleotide introduced by genetic engineering techniquesinto a plasmid or vector derived from a different species is aheterologous polynucleotide. A promoter removed from its native codingsequence and operatively linked to a coding sequence with which it isnot naturally found linked is a heterologous promoter. A site-specificrecombination site that has been cloned into a genome of a virus orviral vector, wherein the genome of the virus does not naturally containit, is a heterologous recombination site. When a polynucleotide with anencoding sequence for a recombinase is used to genetically alter a cellthat does not normally express the recombinase, both the polynucleotideand the recombinase are heterologous to the cell.

As used throughout this specification and the claims, the term“comprise” and its variants including, “comprises”, “comprising”, amongother variants, is inclusive of other components, elements, integers,steps and the like. The term “consists of” or “consisting of” areexclusive of other components, elements, integers, steps and the like.

I. The Simian Adenovirus Sequences

The invention provides nucleic acid sequences and amino acid sequencesof simian Adenovirus 39 (SAdV-39), SAdV-25.2, -26, -30, -37 or -38 whichis isolated from the other material with which they are associated innature.

A. Nucleic Acid Sequences

The SAdV-39 nucleic acid sequences provided herein include nucleotides 1to 36553 of SEQ ID NO:1. The SAdV-25.2 nucleic acid sequences providedherein include nucleotides 1 to 36629 of SEQ ID NO: 130. The SAdV-26nucleic acid sequences provided herein include nucleotides 1 to 36628 ofSEQ ID NO: 162. The SAdV-30 nucleic acid sequences provided hereininclude nucleotides 1 to 36621 of SEQ ID NO: 98. The SAdV-37 nucleicacid sequences provided herein include nucleotides 1 to 36634 of SEQ IDNO: 33. The SAdV-38 nucleic acid sequences provided herein includenucleotides 1 to 36494 of SEQ ID NO: 65.

See, Sequence Listing, which is incorporated by reference herein. In oneembodiment, the nucleic acid sequences of the invention furtherencompass the strand which is complementary to the sequences of SEQ IDNO: 1, 130, 162, 98, 130, or 65, respectively as well as the RNA andcDNA sequences corresponding to the sequences of the following sequencesand their complementary strands. In another embodiment, the nucleic acidsequences further encompass sequences which are greater than 98.5%identical, and preferably, greater than about 99% identical, to theSequence Listing. Also included in one embodiment, are natural variantsand engineered modifications of the sequences provided in SEQ ID NO: 1,130, 162, 98, 130, or 65 and their complementary strands. Suchmodifications include, for example, labels that are known in the art,methylation, and substitution of one or more of the naturally occurringnucleotides with a degenerate nucleotide.

TABLE 1 NUCLEIC ACID REGIONS SAdV-39 SAdV-30 SAdV-25.2 SAdV-26 SAdV-37SAdV-38 ORF ORF ORF ORF ORF ORF SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID Regions NO: 1 NO: 98 NO: 130 NO: 162 NO: 33 NO: 65 ITR 1 . . . 126 1. . . 126 1 . . . 126 1 . . . 131 1 . . . 127 1 . . . 130 E1a 13S JoinJoin Join Join Join Join 12S 576 . . . 1143, 576 . . . 1143, 576 . . .1143, 576 . . . 1140, 577 . . . 1144, 577 . . . 1144, 9S 1228 . . . 14331229 . . . 1434 1229 . . . 1434 1236 . . . 1441 1230 . . . 1435 1230 . .. 1435 E1b Small 1600 . . . 2175 1601 . . . 2173 1600 . . . 2175 1603 .. . 2163 1602 . . . 2174 1601 . . . 2176 T/19K Large 1905 . . . 34161906 . . . 3414 1905 . . . 3416 1908 . . . 3404 1907 . . . 3415 1906 . .. 3417 T/55K IX 3504 . . . 3929 3452 . . . 3922 3501 . . . 3926 3492 . .. 3917 3453 . . . 3923 3505 . . . 3930 E2b pTP Complement ComplementComplement Complement Complement Complement (8474 . . . 10408, (8465 . .. 10399, (8467 . . . 10398, (8465 . . . 10399, (8467 . . . 10400, (8473. . . 10410, 13862 . . . 13870) 13835 . . . 13843) 13826 . . . 13834)13834 . . . 13842) 13836 . . . 13844) 13865 . . . 13873) Poly-Complement Complement Complement Complement Complement Complement merase(5097 . . . 8672, (5091 . . . 8663, (5093 . . . 8665, (5085 . . . 8663,(5092 . . . 8664, (5099 . . . 8671, 13862 . . . 13870) 13835 . . .13843) 13826 . . . 13843) 13834 . . . 13842) 13836 . . . 13844) 13865 .. . 13873) IVa2 Complement Complement Complement Complement ComplementComplement (3994 . . . 5324, (3988 . . . 5318, (3990 . . . 5320, (3982 .. . 5312, (3989 . . . 5319, (3996 . . . 5326, 5603 . . . 5615) 5597 . .. 5609) 5599 . . . 5611) 5591 . . . 5603) 5598 . . . 5610 5605 . . .5617) L1 52/55D 10859 . . . 12037 10826 . . . 12001 10833 . . . 1201710612 . . . 12003 10827 . . . 12002 10871 . . . 12046 IIIa 12064 . . .13833 12028 . . . 13806 12044 . . . 13792 12030 . . . 13805 12029 . . .13807 12073 . . . 13830 L2 Penton 13915 . . . 15510 13888 . . . 1548613874 . . . 15466 13884 . . . 15521 13889 . . . 15514 13913 . . . 15529VII 15517 . . . 16095 15493 . . . 16071 15473 . . . 16054 15528 . . .16106 15521 . . . 16099 15536 . . . 16117 V 16140 . . . 17180 16116 . .. 17153 16102 . . . 17145 16151 . . . 17167 16144 . . . 17181 16165 . .. 17211 pX 17208 . . . 17438 17177 . . . 17407 17173 . . . 17403 17195 .. . 17428 17209 . . . 17439 17239 . . . 17469 L3 VI 17473 . . . 1824917442 . . . 18218 17478 . . . 18209 17461 . . . 18234 17512 . . . 1823417542 . . . 18261 Hexon 18359 . . . 21178 18328 . . . 21141 18315 . . .21113 18344 . . . 21154 18328 . . . 21153 18357 . . . 21146 Endo- 21202. . . 21825 21160 . . . 21786 21136 . . . 21759 21176 . . . 21802 21172. . . 21798 21171 . . . 21791 protease E2a DBP Complement ComplementComplement Complement Complement Complement (21910 . . . 23445) (21871 .. . 23403) (21845 . . . 23377) (21885 . . . 23423) (21883 . . . 23418)(21869 . . . 23404) L4 100 kD 23468 . . . 25870 23426 . . . 25828 23400. . . 25790 23449 . . . 25854 23441 . . . 25840 23430 . . . 25814 33 kDJoin Join Join Join Join Join homolog 25587 . . . 25917, 25548 . . .25875, 25510 . . . 25837, 25574 . . . 25901, 26611 . . . 26940, 25534 .. . 25861, 26087 . . . 26415 26045 . . . 26379 26007 . . . 26350 26071 .. . 26399 27215 . . . 27529 26031 . . . 26377 22 kD 25587 . . . 2615025548 . . . 26111 25510 . . . 26079 25574 . . . 26134 25560 . . . 2612325534 . . . 26103 VIII 26484 . . . 27164 26451 . . . 27131 26425 . . .27105 26482 . . . 27162 26463 . . . 27143 26452 . . . .27132 E3 12.5K27168 . . . 27485 27135 . . . 27452 27109 . . . 27426 27166 . . . 2748327147 . . . 27464 27136 . . . 27453 CR1- 27442 . . . 28071 27409 . . .28032 27383 . . . 28009 27440 . . . 28072 27421 . . . 28044 27410 . . .28036 alpha gp19K 28056 . . . 28583 28017 . . . 28544 27994 . . . 2852728057 . . . 28584 28029 . . . 28556 28021 . . . 28548 CR1- 28616 . . .29233 28581 . . . 29264 28560 . . . 29285 28618 . . . 29355 28593 . . .29276 28581 . . . 29198 beta CR1- 29249 . . . 29863 29280 . . . 2988829301 . . . 29906 29371 . . . 29988 29292 . . . 29900 29214 . . . 29822gamma CR1- 29881 . . . 30765 29906 . . . 30769 29924 . . . 30784 30011 .. . 30883 29918 . . . 30781 29840 . . . 30703 delta RID- 30776 . . .31048 30780 . . . 31052 30795 . . . 31067 30895 . . . 31167 30792 . . .31064 30714 . . . 30986 alpha RID-beta 31057 . . . 31482 31061 . . .31494 31076 . . . 31507 31170 . . . 31613 31073 . . . 31504 30995 . . .31423 14.7K 31478 . . . 31882 31488 . . . 31892 31503 . . . 31907 31609. . . 32010 31500 . . . 31904 31419 . . . 31823 L5 Fiber 31997 . . .33463 32189 . . . 33523 32207 . . . 33535 32264 . . . 33538 32201 . . .33535 32096 . . . 33370 E4 Orf 6/7 Complement Complement ComplementComplement Complement Complement (33567 . . . 33815, (33628 . . . 33876,(33632 . . . 33880, (33635 . . . 33883, (33640 . . . 33888, (33485 . . ..33733, 34529 . . . 34708) 34623 . . . 34772) 34603 . . . 34782) 34630 .. . 34779) 34602 . . . 34784) 34465 . . . 34635) Orf 6 ComplementComplement Complement Complement Complement Complement (33815 . . .34708) (33876 . . . 34772) (33880 . . . 34782) (33883 . . . 34779)(33888 . . . 34784) (33733 . . . 34635) Orf 4 Complement ComplementComplement Complement Complement Complement (34617 . . . 34979) (34678 .. . 35043) (34691 . . . 35053) (34685 . . . 35050) (34763 . . . 35055)(34544 . . . 34906) Orf 3 Complement Complement Complement ComplementComplement Complement (34992 . . . 35342) (35055 . . . 35405) (35066 . .. 35416) (35062 . . . 35412) (35067 . . . 35417) (34919 . . . 35269) Orf2 Complement Complement Complement Complement Complement Complement(35342 . . . 35728) (35405 . . . 35791) (35416 . . . 35802) (35412 . . .35798) (35417 . . . 35803) (35269 . . . 35655) Orf1 ComplementComplement Complement Complement Complement Complement (35772 . . .36143) (35844 . . . 36215) (35846 . . . 36217) (35851 . . . 36222)(35856 . . . 36227) (35699 . . . 36070) ITR Complement ComplementComplement Complement Complement Complement (36428 . . . 36553) (36496 .. . 36621) (36504 . . . 36629) (36498 . . . 36628) (36508 . . . 36634)(36365 . . . 36494)

In one embodiment, fragments of the sequences of SAdV-39, SAdV-25.2,-26, -30, -37 or -38, and their complementary strand, cDNA and RNAcomplementary thereto are provided. Suitable fragments are at least 15nucleotides in length, and encompass functional fragments, i.e.,fragments which are of biological interest. For example, a functionalfragment can express a desired adenoviral product or may be useful inproduction of recombinant viral vectors. Such fragments include the genesequences and fragments listed in the tables herein. The tables providethe transcript regions and open reading frames in the SAdV-39,SAdV-25.2, -26, -30, -37 or -38 sequences. For certain genes, thetranscripts and open reading frames (ORFs) are located on the strandcomplementary to that presented in SEQ ID NO: 1, 130, 162, 98, 130, or65. See, e.g., E2b, E4 and E2a. The calculated molecular weights of theencoded proteins are also shown. Note that the E1a open reading frame ofSAdV-39, SAdV-25.2, -26, -30, -37 or -38 and the E2b open reading framecontain internal splice sites. These splice sites are noted in the tableabove.

The SAdV-39, SAdV-25.2, -26, -30, -37 or -38 adenoviral nucleic acidsequences are useful as therapeutic agents and in construction of avariety of vector systems and host cells. As used herein, a vectorincludes any suitable nucleic acid molecule including, naked DNA, aplasmid, a virus, a cosmid, or an episome. These sequences and productsmay be used alone or in combination with other adenoviral sequences orfragments, or in combination with elements from other adenoviral ornon-adenoviral sequences. The SAdV-39, SAdV-25.2, -26, -30, -37 or -38sequences are also useful as antisense delivery vectors, gene therapyvectors, or vaccine vectors. Thus, further provided are nucleic acidmolecules, gene delivery vectors, and host cells which contain theSAdV-39, SAdV-25.2, -26, -30, -37 or -38 sequences.

For example, the invention encompasses a nucleic acid moleculecontaining simian Ad ITR sequences of the invention. In another example,the invention provides a nucleic acid molecule containing simian Adsequences of the invention encoding a desired Ad gene product. Stillother nucleic acid molecule constructed using the sequences of theinvention will be readily apparent to one of skill in the art, in viewof the information provided herein.

In one embodiment, the simian Ad gene regions identified herein may beused in a variety of vectors for delivery of a heterologous molecule toa cell. For example, vectors are generated for expression of anadenoviral capsid protein (or fragment thereof) for purposes ofgenerating a viral vector in a packaging host cell. Such vectors may bedesigned for expression in trans. Alternatively, such vectors aredesigned to provide cells which stably contain sequences which expressdesired adenoviral functions, e.g., one or more of E1a, E1 b, theterminal repeat sequences, E2a, E2b, E4, E4ORF6 region.

In addition, the adenoviral gene sequences and fragments thereof areuseful for providing the helper functions necessary for production ofhelper-dependent viruses (e.g., adenoviral vectors deleted of essentialfunctions, or adeno-associated viruses (AAV)). For such productionmethods, the SAdV-39, SAdV-25.2, -26, -30, -37 or -38 sequences can beutilized in such a method in a manner similar to those described for thehuman Ad. However, due to the differences in sequences between theSAdV-39, SAdV-25.2, -26, -30, -37 or -38 sequences and those of humanAd, the use of the SAdV-39, SAdV-25.2, -26, -30, -37 or -38 sequencesgreatly minimize or eliminate the possibility of homologousrecombination with helper functions in a host cell carrying human Ad E1functions, e.g., 293 cells, which may produce infectious adenoviralcontaminants during rAAV production.

Methods of producing rAAV using adenoviral helper functions have beendescribed at length in the literature with human adenoviral serotypes.See, e.g., U.S. Pat. No. 6,258,595 and the references cited therein.See, also, U.S. Pat. No. 5,871,982; WO 99/14354; WO 99/15685; WO99/47691. These methods may also be used in production of non-humanserotype AAV, including non-human primate AAV serotypes. The SAdV-39,SAdV-25.2, -26, -30, -37 or -38 sequences which provide the necessaryhelper functions (e.g., E1a, E1b, E2a and/or E4 ORF6) can beparticularly useful in providing the necessary adenoviral function whileminimizing or eliminating the possibility of recombination with anyother adenoviruses present in the rAAV-packaging cell which aretypically of human origin. Thus, selected genes or open reading framesof the SAdV-39, SAdV-25.2, -26, -30, -37 or -38 sequences may beutilized in these rAAV production methods.

Alternatively, recombinant SAdV-39, SAdV-25.2, -26, -30, -37 or -38vectors may be utilized in these methods. Such recombinant adenoviralsimian vectors may include, e.g., a hybrid chimp Ad/AAV in which chimpAd sequences flank a rAAV expression cassette composed of, e.g., AAV 3′and/or 5′ ITRs and a transgene under the control of regulatory sequenceswhich control its expression. One of skill in the art will recognizethat still other simian adenoviral vectors and/or SAdV-39, SAdV-25.2,-26, -30, 37 or -38 gene sequences will be useful for production of rAAVand other viruses dependent upon adenoviral helper.

In still another embodiment, nucleic acid molecules are designed fordelivery and expression of selected adenoviral gene products in a hostcell to achieve a desired physiologic effect. For example, a nucleicacid molecule containing sequences encoding an SAdV-39, SAdV-25.2, -26,-30, -37 or -38 E1a protein may be delivered to a subject for use as acancer therapeutic. Optionally, such a molecule is formulated in alipid-based carrier and preferentially targets cancer cells. Such aformulation may be combined with other cancer therapeutics (e.g.,cisplatin, taxol, or the like). Still other uses for the adenoviralsequences provided herein will be readily apparent to one of skill inthe art.

In addition, one of skill in the art will readily understand that theSAdV-39, SAdV-25.2, -26, -30, -37 or -38 sequences can be readilyadapted for use for a variety of viral and non-viral vector systems forin vitro, ex vivo or in vivo delivery of therapeutic and immunogenicmolecules. For example, the SAdV-39, SAdV-25.2, -26, -30, -37 or -38simian Ad sequences can be utilized in a variety of rAd and non-rAdvector systems. Such vectors systems may include, e.g., plasmids,lentiviruses, retroviruses, poxviruses, vaccinia viruses, andadeno-associated viral systems, among others. Selection of these vectorsystems is not a limitation of the present invention.

The invention further provides molecules useful for production of thesimian and simian-derived proteins of the invention. Such moleculeswhich carry polynucleotides including the simian Ad DNA sequences of theinvention can be in the form of naked DNA, a plasmid, a virus or anyother genetic element.

B. SAdV-39, SAdV-25.2, -26, -30, -37 or -38 Adenoviral Proteins

Gene products of the SAdV-39, SAdV-25.2, -26, -30, -37 or -38adenovirus, such as proteins, enzymes, and fragments thereof, which areencoded by the adenoviral nucleic acids described herein are provided.Further encompassed are SAdV-39, SAdV-25.2, -26, -30, -37 or -38proteins, enzymes, and fragments thereof, having the amino acidsequences encoded by these nucleic acid sequences which are generated byother methods. Such proteins include those encoded by the open readingframes identified in the table above, the proteins identified in theTables below with reference to SEQ ID NO, which are provided in theSequence Listing, and fragments thereof of the proteins andpolypeptides.

PROTEIN SEQUENCES SAdV-39 SAdV-30 SAdV-25.2 SAdV-37 SAdV-38 SAdV-26Regions SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ IDNO: E1a 13S 30 127 159 62 95 191 12S 9S E1b Small T/19K 24 120 153 56 89185 Large T/55K 2 99 131 34 66 163 IX 3 100 132 35 67 164 L1 52/55D 4101 133 36 68 165 IIIa 5 102 134 37 69 166 L2 Penton 6 103 135 38 70 167VII 7 104 136 39 71 168 V 8 105 137 40 72 169 pX 9 106 138 41 73 170 L3VI 10 107 139 42 74 171 Hexon 11 108 140 43 75 172 Endoprotease 12 109141 44 76 173 L4 100 kD 13 110 142 45 77 174 33 kD homolog 32 129 161 6497 193 22 kD 26 122 155 58 91 187 VIII 14 111 143 46 78 175 E3 12.5k 15123 144 47 79 176 CR1-alpha 27 112 156 59 92 188 gp19K 16 124 145 48 87177 CR1-beta 17 113 146 49 80 178 CR1-gamma 18 114 147 50 81 179CR1-delta 19 115 148 51 82 180 RID-alpha 20 116 149 52 83 181 RID-beta21 117 150 53 93 182 14.7K 28 125 158 60 84 189 L5 Fiber 22 118 151 5485 183

Thus, in one aspect, unique simian adenoviral proteins which aresubstantially pure, i.e., are free of other viral and proteinaceousproteins are provided. Preferably, these proteins are at least 10%homogeneous, more preferably 60% homogeneous, and most preferably 95%homogeneous.

In one embodiment, unique simian-derived capsid proteins are provided.As used herein, a simian-derived capsid protein includes any adenoviralcapsid protein that contains a SAdV-39, SAdV-25.2, -26, -30, -37 or -38capsid protein or a fragment thereof, as defined above, including,without limitation, chimeric capsid proteins, fusion proteins,artificial capsid proteins, synthetic capsid proteins, and recombinantcapsid proteins, without limitation to means of generating theseproteins.

Suitably, these simian-derived capsid proteins contain one or moreSAdV-39, SAdV-25.2, -26, -30, -37 or -38 regions or fragments thereof(e.g., a hexon, penton, fiber, or fragment thereof) in combination withcapsid regions or fragments thereof of different adenoviral serotypes,or modified simian capsid proteins or fragments, as described herein. A“modification of a capsid protein associated with altered tropism” asused herein includes an altered capsid protein, i.e., a penton, hexon orfiber protein region, or fragment thereof, such as the knob domain ofthe fiber region, or a polynucleotide encoding same, such thatspecificity is altered. The simian-derived capsid may be constructedwith one or more of the simian Ad of the invention or another Adserotype which may be of human or non-human origin. Such Ad may beobtained from a variety of sources including the ATCC, commercial andacademic sources, or the sequences of the Ad may be obtained fromGenBank or other suitable sources.

The amino acid sequences of the penton proteins of SAdV-39 [SEQ ID NO:6], SAdV-25.2 [SEQ ID NO:135], -26 [SEQ ID NO:167], -30 [SEQ ID NO:103],-37 [SEQ ID NO: 38] or -38 [SEQ ID NO: 70] are provided. Suitably, thispenton protein, or unique fragments thereof, may be utilized for avariety of purposes. Examples of suitable fragments include the pentonhaving N-terminal and/or C-terminal truncations of about 50, 100, 150,or 200 amino acids, based upon the amino acid numbering provided aboveand in SEQ ID NO:6, 103, 135, 38, 70, 167 or 70. Other suitablefragments include shorter internal, C-terminal, or N-terminal fragments.Further, the penton protein may be modified for a variety of purposesknown to those of skill in the art.

Also provided are the amino acid sequences of the hexon protein ofSAdV-39 [SEQ ID NO: 11], SAdV-25.2 [SEQ ID NO:140], -26 [SEQ ID NO:172], -30 [SEQ ID NO: 108], -37 [SEQ ID NO: 43] or -38 [SEQ ID NO: 75].Suitably, this hexon protein, or unique fragments thereof, may beutilized for a variety of purposes. Examples of suitable fragmentsinclude the hexon having N-terminal and/or C-terminal truncations ofabout 50, 100, 150, 200, 300, 400, or 500 amino acids, based upon theamino acid numbering provided above and in SEQ ID NO: 11, 140, 172, 108,43, or 75. Other suitable fragments include shorter internal,C-terminal, or N-terminal fragments. For example, one suitable fragmentthe loop region (domain) of the hexon protein, designated DE1 and FG1,or a hypervariable region thereof. Such fragments include the regionsspanning amino acid residues about 125 to 443; about 138 to 441, orsmaller fragments, such as those spanning about residue 138 to residue163; about 170 to about 176; about 195 to about 203; about 233 to about246; about 253 to about 374; about 287 to about 297; and about 404 toabout 430 of the simian hexon proteins, with reference to SEQ ID NO: 11,140, 172, 108, 43, or 75. Other suitable fragments may be readilyidentified by one of skill in the art. Further, the hexon protein may bemodified for a variety of purposes known to those of skill in the art.Because the hexon protein is the determinant for serotype of anadenovirus, such artificial hexon proteins would result in adenoviruseshaving artificial serotypes. Other artificial capsid proteins can alsobe constructed using the chimp Ad penton sequences and/or fibersequences of the invention and/or fragments thereof.

In one embodiment, an adenovirus having an altered hexon proteinutilizing the sequences of a SAdV-39, SAdV-25.2, -26, -30, -37 or -38hexon protein may be generated. One suitable method for altering hexonproteins is described in U.S. Pat. No. 5,922,315, which is incorporatedby reference. In this method, at least one loop region of the adenovirushexon is changed with at least one loop region of another adenovirusserotype. Thus, at least one loop region of such an altered adenovirushexon protein is a simian Ad hexon loop region of SAdV-39. In oneembodiment, a loop region of the SAdV-39, SAdV-25.2, -26, -30, -37 or-38 hexon protein is replaced by a loop region from another adenovirusserotype. In another embodiment, the loop region of the SAdV-39,SAdV-25.2, -26, -30, -37 or -38 hexon is used to replace a loop regionfrom another adenovirus serotype. Suitable adenovirus serotypes may bereadily selected from among human and non-human serotypes, as describedherein. The selection of a suitable serotype is not a limitation of thepresent invention. Still other uses for the SAdV-39, SAdV-25.2, -26,-30, -37 or -38 hexon protein sequences will be readily apparent tothose of skill in the art.

The amino acid sequences of the fiber proteins of SAdV-39 [SEQ IDNO:22], SAdV-25.2 [SEQ ID NO: 151], -26 [SEQ ID NO: 183], -30 [SEQ IDNO: 118], -37 [SEQ ID NO: 54] or -38 [SEQ ID NO: 85] are provided.Suitably, this fiber protein, or unique fragments thereof, may beutilized for a variety of purposes. One suitable fragment is the fiberknob, located within SEQ ID NO: 22, 151, 183, 119, 54 or 85. Examples ofother suitable fragments include the fiber having N-terminal and/orC-terminal truncations of about 50, 100, 150, or 200 amino acids, basedupon the amino acid numbering provided in SEQ ID NO: 22, 151, 183, 119,54 or 85. Still other suitable fragments include internal fragments.Further, the fiber protein may be modified using a variety of techniquesknown to those of skill in the art.

Unique fragments of the proteins of the SAdV-39, SAdV-25.2, -26, -30,-37 or -38 are at least 8 amino acids in length. However, fragments ofother desired lengths can be readily utilized. In addition,modifications as may be introduced to enhance yield and/or expression ofa SAdV39, SAdV-25.2, -26, -30, -37 or -38 gene product, e.g.,construction of a fusion molecule in which all or a fragment of theSAdV39, SAdV-25.2, -26, -30, -37 or -38 gene product is fused (eitherdirectly or via a linker) with a fusion partner to enhance are providedherein. Other suitable modifications include, without limitation,truncation of a coding region (e.g., a protein or enzyme) to eliminate apre- or pro-protein ordinarily cleaved and to provide the mature proteinor enzyme and/or mutation of a coding region to provide a secretablegene product. Still other modifications will be readily apparent to oneof skill in the art. Further encompassed are proteins having at leastabout 99% identity to the SAdV39, SAdV-25.2, -26, -30, -37 or -38proteins provided herein.

As described herein, vectors of the invention containing the adenoviralcapsid proteins of SAdV-39, SAdV-25.2, -26, -30, -37 or -38 areparticularly well suited for use in applications in which theneutralizing antibodies diminish the effectiveness of other Ad serotypebased vectors, as well as other viral vectors. The rAd vectors areparticularly advantageous in readministration for repeat gene therapy orfor boosting immune response (vaccine titers).

Under certain circumstances, it may be desirable to use one or more ofthe SAdV39, SAdV-25.2, -26, -30, -37 or -38 gene products (e.g., acapsid protein or a fragment thereof) to generate an antibody. The term“an antibody,” as used herein, refers to an immunoglobulin moleculewhich is able to specifically bind to an epitope. The antibodies mayexist in a variety of forms including, for example, high affinitypolyclonal antibodies, monoclonal antibodies, synthetic antibodies,chimeric antibodies, recombinant antibodies and humanized antibodies.Such antibodies originate from immunoglobulin classes IgG, IgM, IgA, IgDand IgE.

Such antibodies may be generated using any of a number of methods knowin the art. Suitable antibodies may be generated by well-knownconventional techniques, e.g., Kohler and Milstein and the many knownmodifications thereof. Similarly desirable high titer antibodies aregenerated by applying known recombinant techniques to the monoclonal orpolyclonal antibodies developed to these antigens [see, e.g., PCT PatentApplication No. PCT/GB85/00392; British Patent Application PublicationNo. GB2188638A; Amit et al., 1986 Science, 233:747-753; Queen et al.,1989 Proc. Natl. Acad. Sci. USA, 86:10029-10033; PCT Patent ApplicationNo. PCT/WO9007861; and Riechmann et al., Nature, 332:323-327 (1988);Huse et al, 1988a Science, 246:1275-1281]. Alternatively, antibodies canbe produced by manipulating the complementarity determining regions ofanimal or human antibodies to the antigen of this invention. See, e.g.,E. Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4,The Handbook of Experimental Pharmacology, Vol. 113, The Pharmacology ofMonoclonal Antibodies, Springer-Verlag (June, 1994); Harlow et al.,1999, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, NY; Harlow et al., 1989, Antibodies: A LaboratoryManual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl.Acad. Sci. USA 85:5879-5883; and Bird et al., 1988, Science 242:423-437.Further provided by the present invention are anti-idiotype antibodies(Ab2) and anti-anti-idiotype antibodies (Ab3). See, e.g., M. Wettendorffet al., “Modulation of anti-tumor immunity by anti-idiotypicantibodies.” In Idiotypic Network and Diseases, ed. by J. Cerny and J.Hiernaux, 1990 J. Am. Soc. Microbiol., Washington D.C.: pp. 203-229].These anti-idiotype and anti-anti-idiotype antibodies are produced usingtechniques well known to those of skill in the art. These antibodies maybe used for a variety of purposes, including diagnostic and clinicalmethods and kits.

Under certain circumstances, it may be desirable to introduce adetectable label or a tag onto a SAdV39, SAdV-25.2, -26, -30, -37 or -38gene product, antibody or other construct of the invention. As usedherein, a detectable label is a molecule which is capable, alone or uponinteraction with another molecule, of providing a detectable signal.Most desirably, the label is detectable visually, e.g. by fluorescence,for ready use in immunohistochemical analyses or immunofluorescentmicroscopy. For example, suitable labels include fluoresceinisothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC),coriphosphine-O (CPO) or tandem dyes, PE-cyanin-5 (PC5), and PE-TexasRed (ECD). All of these fluorescent dyes are commercially available, andtheir uses known to the art. Other useful labels include a colloidalgold label. Still other useful labels include radioactive compounds orelements. Additionally, labels include a variety of enzyme systems thatoperate to reveal a colorimetric signal in an assay, e.g., glucoseoxidase (which uses glucose as a substrate) releases peroxide as aproduct which in the presence of peroxidase and a hydrogen donor such astetramethyl benzidine (TMB) produces an oxidized TMB that is seen as ablue color. Other examples include horseradish peroxidase (HRP),alkaline phosphatase (AP), and hexokinase in conjunction withglucose-6-phosphate dehydrogenase which reacts with ATP, glucose, andNAD+ to yield, among other products, NADH that is detected as increasedabsorbance at 340 nm wavelength.

Other label systems that are utilized in the methods described hereinare detectable by other means, e.g., colored latex microparticles [BangsLaboratories, Indiana] in which a dye is embedded are used in place ofenzymes to form conjugates with the target sequences to provide a visualsignal indicative of the presence of the resulting complex in applicableassays.

Methods for coupling or associating the label with a desired moleculeare similarly conventional and known to those of skill in the art. Knownmethods of label attachment are described [see, for example, Handbook ofFluorescent probes and Research Chemicals, 6th Ed., R. P. M. Haugland,Molecular Probes, Inc., Eugene, Oreg., 1996; Pierce Catalog andHandbook, Life Science and Analytical Research Products, Pierce ChemicalCompany, Rockford, Ill., 1994/1995]. Thus, selection of the label andcoupling methods do not limit this invention.

The sequences, proteins, and fragments of SAdV-39, SAdV-25.2, -26, -30,-37 or -38 may be produced by any suitable means, including recombinantproduction, chemical synthesis, or other synthetic means. Suitableproduction techniques are well known to those of skill in the art. See,e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Press (Cold Spring Harbor, N.Y.). Alternatively, peptidescan also be synthesized by the well known solid phase peptide synthesismethods (Merrifield, J. Am. Chem. Soc., 85:2149 (1962); Stewart andYoung, Solid Phase Peptide Synthesis (Freeman, San Francisco, 1969) pp.27-62). These and other suitable production methods are within theknowledge of those of skill in the art and are not a limitation of thepresent invention.

In addition, one of skill in the art will readily understand that theSAdV-39, SAdV-25.2, -26, -30, -37 or -38 sequences can be readilyadapted for use for a variety of viral and non-viral vector systems forin vitro, ex vivo or in vivo delivery of therapeutic and immunogenicmolecules. For example, in one embodiment, the simian Ad capsid proteinsand other simian adenovirus proteins described herein are used fornon-viral, protein-based delivery of genes, proteins, and otherdesirable diagnostic, therapeutic and immunogenic molecules. In one suchembodiment, a protein of the invention is linked, directly orindirectly, to a molecule for targeting to cells with a receptor foradenoviruses. Preferably, a capsid protein such as a hexon, penton,fiber or a fragment thereof having a ligand for a cell surface receptoris selected for such targeting. Suitable molecules for delivery areselected from among the therapeutic molecules described herein and theirgene products. A variety of linkers including, lipids, polyLys, and thelike may be utilized as linkers. For example, the simian penton proteinmay be readily utilized for such a purpose by production of a fusionprotein using the simian penton sequences in a manner analogous to thatdescribed in Medina-Kauwe L K, et al, Gene Ther. 2001 May; 8(10):795-803and Medina-Kauwe L K, et al, Gene Ther. 2001 December; 8(23): 1753-1761.Alternatively, the amino acid sequences of simian Ad protein IX may beutilized for targeting vectors to a cell surface receptor, as describedin US Patent Appln 20010047081. Suitable ligands include a CD40 antigen,an RGD-containing or polylysine-containing sequence, and the like. Stillother simian Ad proteins, including, e.g., the hexon protein and/or thefiber protein, may be used for used for these and similar purposes.

Still other SAdV-39, SAdV-25.2, -26, -30, -37 or -38 adenoviral proteinsmay be used as alone, or in combination with other adenoviral protein,for a variety of purposes which will be readily apparent to one of skillin the art. In addition, still other uses for the SAdV adenoviralproteins will be readily apparent to one of skill in the art.

II. Recombinant Adenoviral Vectors

The compositions described herein include vectors that deliver aheterologous molecule to cells, either for therapeutic or vaccinepurposes. As used herein, a vector may include any genetic elementincluding, without limitation, naked DNA, a phage, transposon, cosmid,episome, plasmid, or a virus. Such vectors contain simian adenovirus DNAof SAdV39, SAdV-25.2, -26, -30, -37 or -38 and a minigene. By “minigene”or “expression cassette” is meant the combination of a selectedheterologous gene and the other regulatory elements necessary to drivetranslation, transcription and/or expression of the gene product in ahost cell.

Typically, a SAdV-39, SAdV-25.2, -26, -30, -37 or -38-derived adenoviralvector is designed such that the minigene is located in a nucleic acidmolecule which contains other adenoviral sequences in the region nativeto a selected adenoviral gene. The minigene may be inserted into anexisting gene region to disrupt the function of that region, if desired.Alternatively, the minigene may be inserted into the site of a partiallyor fully deleted adenoviral gene. For example, the minigene may belocated in the site of such as the site of a functional E1 deletion orfunctional E3 deletion, among others that may be selected. The term“functionally deleted” or “functional deletion” means that a sufficientamount of the gene region is removed or otherwise damaged, e.g., bymutation or modification, so that the gene region is no longer capableof producing functional products of gene expression. If desired, theentire gene region may be removed. Other suitable sites for genedisruption or deletion are discussed elsewhere in the application.

For example, for a production vector useful for generation of arecombinant virus, the vector may contain the minigene and either the 5′end of the adenoviral genome or the 3′ end of the adenoviral genome, orboth the 5′ and 3′ ends of the adenoviral genome. The 5′ end of theadenoviral genome contains the 5′ cis-elements necessary for packagingand replication; i.e., the 5′ inverted terminal repeat (ITR) sequences(which function as origins of replication) and the native 5′ packagingenhancer domains (that contain sequences necessary for packaging linearAd genomes and enhancer elements for the E1 promoter). The 3′ end of theadenoviral genome includes the 3′ cis-elements (including the ITRs)necessary for packaging and encapsidation. Suitably, a recombinantadenovirus contains both 5′ and 3′ adenoviral cis-elements and theminigene is located between the 5′ and 3′ adenoviral sequences. ASAdV-39, SAdV-25.2, -26, -30, -37 or -38 based adenoviral vector mayalso contain additional adenoviral sequences.

Suitably, these SAdV-39, SAdV-25.2, -26, -30, -37 or -38 basedadenoviral vectors contain one or more adenoviral elements derived fromthe adenoviral genome of the invention. In one embodiment, the vectorscontain adenoviral ITRs from SAdV39, SAdV-25.2, -26, -30, -37 or -38 andadditional adenoviral sequences from the same adenoviral serotype. Inanother embodiment, the vectors contain adenoviral sequences that arederived from a different adenoviral serotype than that which providesthe ITRs.

As defined herein, a pseudotyped adenovirus refers to an adenovirus inwhich the capsid protein of the adenovirus is from a differentadenovirus than the adenovirus which provides the ITRs.

Further, chimeric or hybrid adenoviruses may be constructed using theadenoviruses described herein using techniques known to those of skillin the art. See, e.g., U.S. Pat. No. 7,291,498.

The selection of the adenoviral source of the ITRs and the source of anyother adenoviral sequences present in vector is not a limitation of thepresent embodiment. A variety of adenovirus strains are available fromthe American Type Culture Collection, Manassas, Va., or available byrequest from a variety of commercial and institutional sources. Further,the sequences of many such strains are available from a variety ofdatabases including, e.g., PubMed and GenBank. Homologous adenovirusvectors prepared from other simian or from human adenoviruses aredescribed in the published literature [see, for example, U.S. Pat. No.5,240,846]. The DNA sequences of a number of adenovirus types areavailable from GenBank, including type Ad5 [GenBank Accession No.M73370]. The adenovirus sequences may be obtained from any knownadenovirus serotype, such as serotypes 2, 3, 4, 7, 12 and 40, andfurther including any of the presently identified human types. Similarlyadenoviruses known to infect non-human animals (e.g., simians) may alsobe employed in the vector constructs of this invention. See, e.g., U.S.Pat. No. 6,083,716.

The viral sequences, helper viruses (if needed), and recombinant viralparticles, and other vector components and sequences employed in theconstruction of the vectors described herein are obtained as describedabove. The DNA sequences of the SAdV39, SAdV-25.2, -26, -30, -37 or -38simian adenovirus sequences of the invention are employed to constructvectors and cell lines useful in the preparation of such vectors.

Modifications of the nucleic acid sequences forming the vectors of thisinvention, including sequence deletions, insertions, and other mutationsmay be generated using standard molecular biological techniques and arewithin the scope of this embodiment.

A. The “Minigene”

The methods employed for the selection of the transgene, the cloning andconstruction of the “minigene” and its insertion into the viral vectorare within the skill in the art given the teachings provided herein.

1. The Transgene

The transgene is a nucleic acid sequence, heterologous to the vectorsequences flanking the transgene, which encodes a polypeptide, protein,or other product, of interest. The nucleic acid coding sequence isoperatively linked to regulatory components in a manner which permitstransgene transcription, translation, and/or expression in a host cell.

The composition of the transgene sequence will depend upon the use towhich the resulting vector will be put. For example, one type oftransgene sequence includes a reporter sequence, which upon expressionproduces a detectable signal. Such reporter sequences include, withoutlimitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ),alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP),chloramphenicol acetyltransferase (CAT), luciferase, membrane boundproteins including, for example, CD2, CD4, CD8, the influenzahemagglutinin protein, and others well known in the art, to which highaffinity antibodies directed thereto exist or can be produced byconventional means, and fusion proteins comprising a membrane boundprotein appropriately fused to an antigen tag domain from, among others,hemagglutinin or Myc. These coding sequences, when associated withregulatory elements which drive their expression, provide signalsdetectable by conventional means, including enzymatic, radiographic,colorimetric, fluorescence or other spectrographic assays, fluorescentactivating cell sorting assays and immunological assays, includingenzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) andimmunohistochemistry. For example, where the marker sequence is the LacZgene, the presence of the vector carrying the signal is detected byassays for beta-galactosidase activity. Where the transgene is GFP orluciferase, the vector carrying the signal may be measured visually bycolor or light production in a luminometer.

In one embodiment, the transgene is a non-marker sequence encoding aproduct which is useful in biology and medicine, such as proteins,peptides, RNA, enzymes, or catalytic RNAs. Desirable RNA moleculesinclude tRNA, dsRNA, ribosomal RNA, catalytic RNAs, and antisense RNAs.One example of a useful RNA sequence is a sequence which extinguishesexpression of a targeted nucleic acid sequence in the treated animal.

The transgene may be used for treatment, e.g., of genetic deficiencies,as a cancer therapeutic or vaccine, for induction of an immune response,and/or for prophylactic vaccine purposes. As used herein, induction ofan immune response refers to the ability of a molecule (e.g., a geneproduct) to induce a T cell and/or a humoral immune response to themolecule. The invention further includes using multiple transgenes,e.g., to correct or ameliorate a condition caused by a multi-subunitprotein. In certain situations, a different transgene may be used toencode each subunit of a protein, or to encode different peptides orproteins. This is desirable when the size of the DNA encoding theprotein subunit is large, e.g., for an immunoglobulin, theplatelet-derived growth factor, or a dystrophin protein. In order forthe cell to produce the multi-subunit protein, a cell is infected withthe recombinant virus containing each of the different subunits.Alternatively, different subunits of a protein may be encoded by thesame transgene. In this case, a single transgene includes the DNAencoding each of the subunits, with the DNA for each subunit separatedby an internal ribozyme entry site (IRES). This is desirable when thesize of the DNA encoding each of the subunits is small, e.g., the totalsize of the DNA encoding the subunits and the IRES is less than fivekilobases. As an alternative to an IRES, the DNA may be separated bysequences encoding a 2A peptide, which self-cleaves in apost-translational event. See, e.g., M. L. Donnelly, et al, J. Gen.Virol., 78(Pt 1):13-21 (January 1997); Furler, S., et al, Gene Ther.,8(11):864-873 (June 2001); Klump H., et al., Gene Ther., 8(10):811-817(May 2001). This 2A peptide is significantly smaller than an IRES,making it well suited for use when space is a limiting factor. However,the selected transgene may encode any biologically active product orother product, e.g., a product desirable for study.

Suitable transgenes may be readily selected by one of skill in the art.The selection of the transgene is not considered to be a limitation ofthis embodiment.

2. Regulatory Elements

In addition to the major elements identified above for the minigene, thevector also includes conventional control elements necessary which areoperably linked to the transgene in a manner that permits itstranscription, translation and/or expression in a cell transfected withthe plasmid vector or infected with the virus produced by the invention.As used herein, “operably linked” sequences include both expressioncontrol sequences that are contiguous with the gene of interest andexpression control sequences that act in trans or at a distance tocontrol the gene of interest.

Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation (polyA) signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhancesecretion of the encoded product.

A great number of expression control sequences, including promoterswhich are native, constitutive, inducible and/or tissue-specific, areknown in the art and may be utilized. Examples of constitutive promotersinclude, without limitation, the retroviral Rous sarcoma virus (RSV) LTRpromoter (optionally with the RSV enhancer), the cytomegalovirus (CMV)promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al,Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductasepromoter, the β-actin promoter, the phosphoglycerol kinase (PGK)promoter, and the EF1α promoter [Invitrogen].

Inducible promoters allow regulation of gene expression and can beregulated by exogenously supplied compounds, environmental factors suchas temperature, or the presence of a specific physiological state, e.g.,acute phase, a particular differentiation state of the cell, or inreplicating cells only. Inducible promoters and inducible systems areavailable from a variety of commercial sources, including, withoutlimitation, Invitrogen, Clontech and Ariad. Many other systems have beendescribed and can be readily selected by one of skill in the art. Forexample, inducible promoters include the zinc-inducible sheepmetallothionine (MT) promoter and the dexamethasone (Dex)-induciblemouse mammary tumor virus (MMTV) promoter. Other inducible systemsinclude the T7 polymerase promoter system [WO 98/10088]; the ecdysoneinsect promoter [No et al, Proc. Natl. Acad. Sci. USA, 93:3346-3351(1996)], the tetracycline-repressible system [Gossen et al, Proc. Natl.Acad. Sci. USA, 89:5547-5551 (1992)], the tetracycline-inducible system[Gossen et al, Science, 378:1766-1769 (1995), see also Harvey et al,Curr. Opin. Chem. Biol., 2:512-518 (1998)]. Other systems include theFK506 dimer, VP16 or p65 using castradiol, diphenol murislerone, theRU486-inducible system [Wang et al, Nat. Biotech., 15:239-243 (1997) andWang et al, Gene Ther., 4:432-441 (1997)] and the rapamycin-induciblesystem [Magari et al, J. Clin. Invest., 100:2865-2872 (1997)]. Theeffectiveness of some inducible promoters increases over time. In suchcases one can enhance the effectiveness of such systems by insertingmultiple repressors in tandem, e.g., TetR linked to a TetR by an IRES.Alternatively, one can wait at least 3 days before screening for thedesired function. One can enhance expression of desired proteins byknown means to enhance the effectiveness of this system. For example,using the Woodchuck Hepatitis Virus Posttranscriptional RegulatoryElement (WPRE).

In another embodiment, the native promoter for the transgene will beused. The native promoter may be preferred when it is desired thatexpression of the transgene should mimic the native expression. Thenative promoter may be used when expression of the transgene must beregulated temporally or developmentally, or in a tissue-specific manner,or in response to specific transcriptional stimuli. In a furtherembodiment, other native expression control elements, such as enhancerelements, polyadenylation sites or Kozak consensus sequences may also beused to mimic the native expression.

Another embodiment of the transgene includes a transgene operably linkedto a tissue-specific promoter. For instance, if expression in skeletalmuscle is desired, a promoter active in muscle should be used. Theseinclude the promoters from genes encoding skeletal β-actin, myosin lightchain 2A, dystrophin, muscle creatine kinase, as well as syntheticmuscle promoters with activities higher than naturally occurringpromoters (see Li et al., Nat. Biotech., 17:241-245 (1999)). Examples ofpromoters that are tissue-specific are known for liver (albumin,Miyatake et al., J. Virol., 71:5124-32 (1997); hepatitis B virus corepromoter, Sandig et al., Gene Ther., 3:1002-9 (1996); alpha-fetoprotein(AFP), Arbuthnot et al., Hum. Gene Ther., 7:1503-14 (1996)), boneosteocalcin (Stein et al., Mol. Biol. Rep., 24:185-96 (1997)); bonesialoprotein (Chen et al., J. Bone Miner. Res., 11:654-64 (1996)),lymphocytes (CD2, Hansal et al., J. Immunol., 161:1063-8 (1998);immunoglobulin heavy chain; T cell receptor chain), neuronal such asneuron-specific enolase (NSE) promoter (Andersen et al., Cell. Mol.Neurobiol., 13:503-15 (1993)), neurofilament light-chain gene (Piccioliet al., Proc. Natl. Acad. Sci. USA, 88:5611-5 (1991)), and theneuron-specific vgf gene (Piccioli et al., Neuron, 15:373-84 (1995)),among others.

Optionally, vectors carrying transgenes encoding therapeutically usefulor immunogenic products may also include selectable markers or reportergenes may include sequences encoding geneticin, hygromicin or purimycinresistance, among others. Such selectable reporters or marker genes(preferably located outside the viral genome to be packaged into a viralparticle) can be used to signal the presence of the plasmids inbacterial cells, such as ampicillin resistance. Other components of thevector may include an origin of replication. Selection of these andother promoters and vector elements are conventional and many suchsequences are available [see, e.g., Sambrook et al, and references citedtherein].

These vectors are generated using the techniques and sequences providedherein, in conjunction with techniques known to those of skill in theart. Such techniques include conventional cloning techniques of cDNAsuch as those described in texts [Sambrook et al, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.],use of overlapping oligonucleotide sequences of the adenovirus genomes,polymerase chain reaction, and any suitable method which provides thedesired nucleotide sequence.

III. Production of the Viral Vector

In one embodiment, the simian adenoviral plasmids (or other vectors) areused to produce adenoviral vectors. In one embodiment, the adenoviralvectors are adenoviral particles which are replication-defective. In oneembodiment, the adenoviral particles are rendered replication-defectiveby deletions in the E1a and/or E1b genes. Alternatively, theadenoviruses are rendered replication-defective by another means,optionally while retaining the E1a and/or E1b genes. The adenoviralvectors can also contain other mutations to the adenoviral genome, e.g.,temperature-sensitive mutations or deletions in other genes. In otherembodiments, it is desirable to retain an intact E1a and/or E1b regionin the adenoviral vectors. Such an intact E1 region may be located inits native location in the adenoviral genome or placed in the site of adeletion in the native adenoviral genome (e.g., in the E3 region).

In the construction of useful simian adenovirus vectors for delivery ofa gene to the human (or other mammalian) cell, a range of adenovirusnucleic acid sequences can be employed in the vectors. For example, allor a portion of the adenovirus delayed early gene E3 may be eliminatedfrom the simian adenovirus sequence which forms a part of therecombinant virus. The function of simian E3 is believed to beirrelevant to the function and production of the recombinant virusparticle. Simian adenovirus vectors may also be constructed having adeletion of at least the ORF6 region of the E4 gene, and more desirablybecause of the redundancy in the function of this region, the entire E4region. Still another vector of this invention contains a deletion inthe delayed early gene E2a. Deletions may also be made in any of thelate genes L1 through L5 of the simian adenovirus genome. Similarly,deletions in the intermediate genes IX and IVa₂ may be useful for somepurposes. Other deletions may be made in the other structural ornon-structural adenovirus genes. The above discussed deletions may beused individually, i.e., an adenovirus sequence for use as describedherein may contain deletions in only a single region. Alternatively,deletions of entire genes or portions thereof effective to destroy theirbiological activity may be used in any combination. For example, in oneexemplary vector, the adenovirus sequence may have deletions of the E1genes and the E4 gene, or of the E1, E2a and E3 genes, or of the E1 andE3 genes, or of E1, E2a and E4 genes, with or without deletion of E3,and so on. As discussed above, such deletions may be used in combinationwith other mutations, such as temperature-sensitive mutations, toachieve a desired result.

An adenoviral vector lacking any essential adenoviral sequences (e.g.,E1a, E1b, E2a, E2b, E4 ORF6, L1, L2, L3, L4 and L5) may be cultured inthe presence of the missing adenoviral gene products which are requiredfor viral infectivity and propagation of an adenoviral particle. Thesehelper functions may be provided by culturing the adenoviral vector inthe presence of one or more helper constructs (e.g., a plasmid or virus)or a packaging host cell. See, for example, the techniques described forpreparation of a “minimal” human Ad vector in International PatentApplication WO96/13597, published May 9, 1996, and incorporated hereinby reference.

1. Helper Viruses

Thus, depending upon the simian adenovirus gene content of the viralvectors employed to carry the minigene, a helper adenovirus ornon-replicating virus fragment may be necessary to provide sufficientsimian adenovirus gene sequences necessary to produce an infectiverecombinant viral particle containing the minigene. Useful helperviruses contain selected adenovirus gene sequences not present in theadenovirus vector construct and/or not expressed by the packaging cellline in which the vector is transfected. In one embodiment, the helpervirus is replication-defective and contains a variety of adenovirusgenes in addition to the sequences described above. Such a helper virusis desirably used in combination with an E1-expressing cell line.

Helper viruses may also be formed into poly-cation conjugates asdescribed in Wu et al, J. Biol. Chem., 374:16985-16987 (1989); K. J.Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994). Helpervirus may optionally contain a second reporter minigene. A number ofsuch reporter genes are known to the art. The presence of a reportergene on the helper virus which is different from the transgene on theadenovirus vector allows both the Ad vector and the helper virus to beindependently monitored. This second reporter is used to enableseparation between the resulting recombinant virus and the helper virusupon purification.

2. Complementation Cell Lines

To generate recombinant simian adenoviruses (Ad) deleted in any of thegenes described above, the function of the deleted gene region, ifessential to the replication and infectivity of the virus, must besupplied to the recombinant virus by a helper virus or cell line, i.e.,a complementation or packaging cell line. In many circumstances, a cellline expressing the human E1 can be used to transcomplement the chimp Advector. This is particularly advantageous because, due to the diversitybetween the chimp Ad sequences of the invention and the human AdE1sequences found in currently available packaging cells, the use of thecurrent human E1-containing cells prevents the generation ofreplication-competent adenoviruses during the replication and productionprocess. However, in certain circumstances, it will be desirable toutilize a cell line which expresses the E1 gene products that can beutilized for production of an E1-deleted simian adenovirus. Such celllines have been described. See, e.g., U.S. Pat. No. 6,083,716.

If desired, one may utilize the sequences provided herein to generate apackaging cell or cell line that expresses, at a minimum, the adenovirusE1 gene from SAdV39 under the transcriptional control of a promoter forexpression in a selected parent cell line. Inducible or constitutivepromoters may be employed for this purpose. Examples of such promotersare described in detail elsewhere in this specification. A parent cellis selected for the generation of a novel cell line expressing anydesired SAdV39 gene. Without limitation, such a parent cell line may beHeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No. CCL 185], HEK293, KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75]cells, among others. These cell lines are all available from theAmerican Type Culture Collection, 10801 University Boulevard, Manassas,Va. 20110-2209. Other suitable parent cell lines may be obtained fromother sources.

Such E1-expressing cell lines are useful in the generation ofrecombinant simian adenovirus E1 deleted vectors. Additionally, oralternatively, cell lines that express one or more simian adenoviralgene products, e.g., E1a, E1b, E2a, and/or E4 ORF6, can be constructedusing essentially the same procedures are used in the generation ofrecombinant simian viral vectors. Such cell lines can be utilized totranscomplement adenovirus vectors deleted in the essential genes thatencode those products, or to provide helper functions necessary forpackaging of a helper-dependent virus (e.g., adeno-associated virus).The preparation of a host cell involves techniques such as assembly ofselected DNA sequences. This assembly may be accomplished utilizingconventional techniques. Such techniques include cDNA and genomiccloning, which are well known and are described in Sambrook et al.,cited above, use of overlapping oligonucleotide sequences of theadenovirus genomes, combined with polymerase chain reaction, syntheticmethods, and any other suitable methods which provide the desirednucleotide sequence.

In still another alternative, the essential adenoviral gene products areprovided in trans by the adenoviral vector and/or helper virus. In suchan instance, a suitable host cell can be selected from any biologicalorganism, including prokaryotic (e.g., bacterial) cells, and eukaryoticcells, including, insect cells, yeast cells and mammalian cells.Particularly desirable host cells are selected from among any mammalianspecies, including, without limitation, cells such as A549, WEHI, 3T3,10T1/2, HEK 293 cells or PERC6 (both of which express functionaladenoviral E1) [Fallaux, F J et al, (1998), Hum Gene Ther, 9:1909-1917],Saos, C2C12, L cells, HT1080, HepG2 and primary fibroblast, hepatocyteand myoblast cells derived from mammals including human, monkey, mouse,rat, rabbit, and hamster. The selection of the mammalian speciesproviding the cells is not a limitation of this invention; nor is thetype of mammalian cell, i.e., fibroblast, hepatocyte, tumor cell, etc.

3. Assembly of Viral Particle and Transfection of a Cell Line

Generally, when delivering the vector comprising the minigene bytransfection, the vector is delivered in an amount from about 5 μg toabout 100 μg DNA, and preferably about 10 to about 50 μg DNA to about1×10⁴ cells to about 1×10¹³ cells, and preferably about 10⁵ cells.However, the relative amounts of vector DNA to host cells may beadjusted, taking into consideration such factors as the selected vector,the delivery method and the host cells selected.

The vector may be any vector known in the art or disclosed above,including naked DNA, a plasmid, phage, transposon, cosmids, episomes,viruses, etc. Introduction into the host cell of the vector may beachieved by any means known in the art or as disclosed above, includingtransfection, and infection. One or more of the adenoviral genes may bestably integrated into the genome of the host cell, stably expressed asepisomes, or expressed transiently. The gene products may all beexpressed transiently, on an episome or stably integrated, or some ofthe gene products may be expressed stably while others are expressedtransiently. Furthermore, the promoters for each of the adenoviral genesmay be selected independently from a constitutive promoter, an induciblepromoter or a native adenoviral promoter. The promoters may be regulatedby a specific physiological state of the organism or cell (i.e., by thedifferentiation state or in replicating or quiescent cells) or byexogenously-added factors, for example.

Introduction of the molecules (as plasmids or viruses) into the hostcell may also be accomplished using techniques known to the skilledartisan and as discussed throughout the specification. In preferredembodiment, standard transfection techniques are used, e.g., CaPO₄transfection or electroporation.

Assembly of the selected DNA sequences of the adenovirus (as well as thetransgene and other vector elements into various intermediate plasmids,and the use of the plasmids and vectors to produce a recombinant viralparticle are all achieved using conventional techniques. Such techniquesinclude conventional cloning techniques of cDNA such as those describedin texts [Sambrook et al, cited above], use of overlappingoligonucleotide sequences of the adenovirus genomes, polymerase chainreaction, and any suitable method which provides the desired nucleotidesequence. Standard transfection and co-transfection techniques areemployed, e.g., CaPO₄ precipitation techniques. Other conventionalmethods employed include homologous recombination of the viral genomes,plaquing of viruses in agar overlay, methods of measuring signalgeneration, and the like.

For example, following the construction and assembly of the desiredminigene-containing viral vector, the vector is transfected in vitro inthe presence of a helper virus into the packaging cell line. Homologousrecombination occurs between the helper and the vector sequences, whichpermits the adenovirus-transgene sequences in the vector to bereplicated and packaged into virion capsids, resulting in therecombinant viral vector particles. The current method for producingsuch virus particles is transfection-based. However, the invention isnot limited to such methods.

The resulting recombinant simian adenoviruses are useful in transferringa selected transgene to a selected cell. In in vivo experiments with therecombinant virus grown in the packaging cell lines, the E1-deletedrecombinant simian adenoviral vectors of the invention demonstrateutility in transferring a transgene to a non-simian, preferably a human,cell.

IV. Use of the Recombinant Adenovirus Vectors

The recombinant simian adenovirus -39, SAdV-25.2, -26, -30, -37 or -38based vectors are useful for gene transfer to a human or non-simianveterinary patient in vitro, ex vivo, and in vivo.

The recombinant adenovirus vectors described herein can be used asexpression vectors for the production of the products encoded by theheterologous genes in vitro. For example, the recombinant adenovirusescontaining a gene inserted into the location of an E1 deletion may betransfected into an E1-expressing cell line as described above.Alternatively, replication-competent adenoviruses may be used in anotherselected cell line. The transfected cells are then cultured in theconventional manner, allowing the recombinant adenovirus to express thegene product from the promoter. The gene product may then be recoveredfrom the culture medium by known conventional methods of proteinisolation and recovery from culture.

A SAdV39, SAdV-25.2, -26, -30, -37 or -38-derived recombinant simianadenoviral vector provides an efficient gene transfer vehicle that candeliver a selected transgene to a selected host cell in vivo or ex vivoeven where the organism has neutralizing antibodies to one or more AAVserotypes. In one embodiment, the rAAV and the cells are mixed ex vivo;the infected cells are cultured using conventional methodologies; andthe transduced cells are re-infused into the patient. These compositionsare particularly well suited to gene delivery for therapeutic purposesand for immunization, including inducing protective immunity.

More commonly, the SAdV39, SAdV-25.2, -26, -30, -37 or -38 recombinantadenoviral vectors will be utilized for delivery of therapeutic orimmunogenic molecules, as described below. It will be readily understoodfor both applications, that the recombinant adenoviral vectors of theinvention are particularly well suited for use in regimens involvingrepeat delivery of recombinant adenoviral vectors. Such regimenstypically involve delivery of a series of viral vectors in which theviral capsids are alternated. The viral capsids may be changed for eachsubsequent administration, or after a pre-selected number ofadministrations of a particular serotype capsid (e.g., one, two, three,four or more). Thus, a regimen may involve delivery of a rAd with afirst simian capsid, delivery with a rAd with a second simian capsid,and delivery with a third simian capsid. A variety of other regimenswhich use the Ad capsids of the invention alone, in combination with oneanother, or in combination with other adenoviruses (which are preferablyimmunologically non-crossreactive) will be apparent to those of skill inthe art. Optionally, such a regimen may involve administration of rAdwith capsids of other non-human primate adenoviruses, humanadenoviruses, or artificial sequences such as are described herein. Eachphase of the regimen may involve administration of a series ofinjections (or other delivery routes) with a single Ad capsid followedby a series with another capsid from a different Ad source.Alternatively, the SAdV-39, SAdV-25.2, -26, -30, -37 or -38 vectors maybe utilized in regimens involving other non-adenoviral-mediated deliverysystems, including other viral systems, non-viral delivery systems,protein, peptides, and other biologically active molecules.

The following sections will focus on exemplary molecules which may bedelivered via the adenoviral vectors of the invention.

A. Ad-Mediated Delivery of Therapeutic Molecules

In one embodiment, the above-described recombinant vectors areadministered to humans according to published methods for gene therapy.A simian viral vector bearing the selected transgene may be administeredto a patient, preferably suspended in a biologically compatible solutionor pharmaceutically acceptable delivery vehicle. A suitable vehicleincludes sterile saline. Other aqueous and non-aqueous isotonic sterileinjection solutions and aqueous and non-aqueous sterile suspensionsknown to be pharmaceutically acceptable carriers and well known to thoseof skill in the art may be employed for this purpose.

The simian adenoviral vectors are administered in sufficient amounts totransduce the target cells and to provide sufficient levels of genetransfer and expression to provide a therapeutic benefit without undueadverse or with medically acceptable physiological effects, which can bedetermined by those skilled in the medical arts. Conventional andpharmaceutically acceptable routes of administration include, but arenot limited to, direct delivery to the retina and other intraoculardelivery methods, direct delivery to the liver, inhalation, intranasal,intravenous, intramuscular, intratracheal, subcutaneous, intradermal,rectal, oral and other parenteral routes of administration. Routes ofadministration may be combined, if desired, or adjusted depending uponthe transgene or the condition. The route of administration primarilywill depend on the nature of the condition being treated.

Dosages of the viral vector will depend primarily on factors such as thecondition being treated, the age, weight and health of the patient, andmay thus vary among patients. For example, a therapeutically effectiveadult human or veterinary dosage of the viral vector is generally in therange of from about 100 μL to about 100 mL of a carrier containingconcentrations of from about 1×10⁶ to about 1×10¹⁵ particles, about1×10¹¹ to 1×10¹³ particles, or about 1×10⁹ to 1×10¹² particles virus.Dosages will range depending upon the size of the animal and the routeof administration. For example, a suitable human or veterinary dosage(for about an 80 kg animal) for intramuscular injection is in the rangeof about 1×10⁹ to about 5×10¹² particles per mL, for a single site.Optionally, multiple sites of administration may be delivered. Inanother example, a suitable human or veterinary dosage may be in therange of about 1×10¹¹ to about 1×10¹⁵ particles for an oral formulation.One of skill in the art may adjust these doses, depending the route ofadministration, and the therapeutic or vaccinal application for whichthe recombinant vector is employed. The levels of expression of thetransgene, or for an immunogen, the level of circulating antibody, canbe monitored to determine the frequency of dosage administration. Yetother methods for determining the timing of frequency of administrationwill be readily apparent to one of skill in the art.

An optional method step involves the co-administration to the patient,either concurrently with, or before or after administration of the viralvector, of a suitable amount of a short acting immune modulator. Theselected immune modulator is defined herein as an agent capable ofinhibiting the formation of neutralizing antibodies directed against therecombinant vector of this invention or capable of inhibiting cytolyticT lymphocyte (CTL) elimination of the vector. The immune modulator mayinterfere with the interactions between the T helper subsets (T_(H1) orT_(H2)) and B cells to inhibit neutralizing antibody formation.Alternatively, the immune modulator may inhibit the interaction betweenT_(H1) cells and CTLs to reduce the occurrence of CTL elimination of thevector. A variety of useful immune modulators and dosages for use ofsame are disclosed, for example, in Yang et al., J. Virol., 70(9)(September, 1996); International Patent Application No. WO96/12406,published May 2, 1996; and International Patent Application No.PCT/US96/03035, all incorporated herein by reference.

1. Therapeutic Transgenes

Useful therapeutic products encoded by the transgene include hormonesand growth and differentiation factors including, without limitation,insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH),growth hormone releasing factor (GRF), follicle stimulating hormone(FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG),vascular endothelial growth factor (VEGF), angiopoietins, angiostatin,granulocyte colony stimulating factor (GCSF), erythropoietin (EPO),connective tissue growth factor (CTGF), basic fibroblast growth factor(bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor(EGF), transforming growth factor (TGF), platelet-derived growth factor(PDGF), insulin growth factors I and II (IGF-I and IGF-II), any one ofthe transforming growth factor superfamily, including TGF, activins,inhibins, or any of the bone morphogenic proteins (BMP) BMPs 1-15, anyone of the heregluin/neuregulin/ARIA/neu differentiation factor (NDF)family of growth factors, nerve growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophins NT-3 and NT-4/5, ciliaryneurotrophic factor (CNTF), glial cell line derived neurotrophic factor(GDNF), neurturin, agrin, any one of the family ofsemaphorins/collapsins, netrin-1 and netrin-2, hepatocyte growth factor(HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

Other useful transgene products include proteins that regulate theimmune system including, without limitation, cytokines and lymphokinessuch as thrombopoietin (TPO), interleukins (IL) IL-1 through IL-25(including, e.g., IL-2, IL-4, IL-12 and IL-18), monocyte chemoattractantprotein, leukemia inhibitory factor, granulocyte-macrophage colonystimulating factor, Fas ligand, tumor necrosis factors and, interferons,and, stem cell factor, flk-2/flt3 ligand. Gene products produced by theimmune system are also useful in the invention. These include, withoutlimitation, immunoglobulins IgG, IgM, IgA, IgD and IgE, chimericimmunoglobulins, humanized antibodies, single chain antibodies, T cellreceptors, chimeric T cell receptors, single chain T cell receptors,class I and class II MHC molecules, as well as engineeredimmunoglobulins and MHC molecules. Useful gene products also includecomplement regulatory proteins such as complement regulatory proteins,membrane cofactor protein (MCP), decay accelerating factor (DAF), CR1,CF2 and CD59.

Still other useful gene products include any one of the receptors forthe hormones, growth factors, cytokines, lymphokines, regulatoryproteins and immune system proteins. The invention encompasses receptorsfor cholesterol regulation, including the low density lipoprotein (LDL)receptor, high density lipoprotein (HDL) receptor, the very low densitylipoprotein (VLDL) receptor, and the scavenger receptor. The inventionalso encompasses gene products such as members of the steroid hormonereceptor superfamily including glucocorticoid receptors and estrogenreceptors, Vitamin D receptors and other nuclear receptors. In addition,useful gene products include transcription factors such as jun, fos,max, mad, serum response factor (SRF), AP-1, AP2, myb, MyoD andmyogenin, ETS-box containing proteins, TFE3, E2F, ATF1, ATF2, ATF3,ATF4, ZF5, NFAT, CREB, HNF-4, C/EBP, SP1, CCAAT-box binding proteins,interferon regulation factor (IRF-1), Wilms tumor protein, ETS-bindingprotein, STAT, GATA-box binding proteins, e.g., GATA-3, and the forkheadfamily of winged helix proteins.

Other useful gene products include, carbamoyl synthetase I, ornithinetranscarbamylase, arginosuccinate synthetase, arginosuccinate lyase,arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase,alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase,factor VIII, factor IX, cystathione beta-synthase, branched chainketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionylCoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase,insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase,phosphorylase kinase, glycine decarboxylase, H-protein, T-protein, acystic fibrosis transmembrane regulator (CFTR) sequence, and adystrophin cDNA sequence.

Other useful gene products include non-naturally occurring polypeptides,such as chimeric or hybrid polypeptides having a non-naturally occurringamino acid sequence containing insertions, deletions or amino acidsubstitutions. For example, single-chain engineered immunoglobulinscould be useful in certain immunocompromised patients. Other types ofnon-naturally occurring gene sequences include antisense molecules andcatalytic nucleic acids, such as ribozymes, which could be used toreduce overexpression of a target.

Reduction and/or modulation of expression of a gene are particularlydesirable for treatment of hyperproliferative conditions characterizedby hyperproliferating cells, as are cancers and psoriasis. Targetpolypeptides include those polypeptides which are produced exclusivelyor at higher levels in hyperproliferative cells as compared to normalcells. Target antigens include polypeptides encoded by oncogenes such asmyb, myc, fyn, and the translocation gene bcr/abl, ras, src, P53, neu,trk and EGRF. In addition to oncogene products as target antigens,target polypeptides for anti-cancer treatments and protective regimensinclude variable regions of antibodies made by B cell lymphomas andvariable regions of T cell receptors of T cell lymphomas which, in someembodiments, are also used as target antigens for autoimmune disease.Other tumor-associated polypeptides can be used as target polypeptidessuch as polypeptides which are found at higher levels in tumor cellsincluding the polypeptide recognized by monoclonal antibody 17-1A andfolate binding polypeptides.

Other suitable therapeutic polypeptides and proteins include those whichmay be useful for treating individuals suffering from autoimmunediseases and disorders by conferring a broad based protective immuneresponse against targets that are associated with autoimmunity includingcell receptors and cells which produce self-directed antibodies. T cellmediated autoimmune diseases include Rheumatoid arthritis (RA), multiplesclerosis (MS), Sjögren's syndrome, sarcoidosis, insulin dependentdiabetes mellitus (IDDM), autoimmune thyroiditis, reactive arthritis,ankylosing spondylitis, scleroderma, polymyositis, dermatomyositis,psoriasis, vasculitis, Wegener's granulomatosis, Crohn's disease andulcerative colitis. Each of these diseases is characterized by T cellreceptors (TCRs) that bind to endogenous antigens and initiate theinflammatory cascade associated with autoimmune diseases.

The simian adenoviral vectors of the invention are particularly wellsuited for therapeutic regimens in which multiple adenoviral-mediateddeliveries of transgenes is desired, e.g., in regimens involvingredelivery of the same transgene or in combination regimens involvingdelivery of other transgenes. Such regimens may involve administrationof a SAdV39, SAdV-25.2, -26, -30, -37 or -38 simian adenoviral vector,followed by re-administration with a vector from the same serotypeadenovirus. Particularly desirable regimens involve administration of aSAdV39, SAdV-25.2, -26, -30, -37 or -38 simian adenoviral vector, inwhich the source of the adenoviral capsid sequences of the vectordelivered in the first administration differs from the source ofadenoviral capsid sequences of the viral vector utilized in one or moreof the subsequent administrations. For example, a therapeutic regimeninvolves administration of a SAdV39, SAdV-25.2, -26, -30, -37 or -38vector and repeat administration with one or more adenoviral vectors ofthe same or different serotypes. In another example, a therapeuticregimen involves administration of an adenoviral vector followed byrepeat administration with a SAdV39, SAdV-25.2, -26, -30, -37 or -38vector which has a capsid which differs from the source of the capsid inthe first delivered adenoviral vector, and optionally furtheradministration with another vector which is the same or, preferably,differs from the source of the adenoviral capsid of the vector in theprior administration steps. These regimens are not limited to deliveryof adenoviral vectors constructed using the SAdV39, SAdV-25.2, -26, -30,-37 or -38 simian sequences. Rather, these regimens can readily utilizeother adenoviral sequences, including, without limitation, other simianadenoviral sequences, (e.g., Pan9 or C68, C1, etc), other non-humanprimate adenoviral sequences, or human adenoviral sequences, incombination with one or more of the SAdV39, SAdV-25.2, -26, -30, -37 or-38 vectors. Examples of such simian, other non-human primate and humanadenoviral serotypes are discussed elsewhere in this document. Further,these therapeutic regimens may involve either simultaneous or sequentialdelivery of SAdV39, SAdV-25.2, -26, -30, -37 or -38 adenoviral vectorsin combination with non-adenoviral vectors, non-viral vectors, and/or avariety of other therapeutically useful compounds or molecules. Theinvention is not limited to these therapeutic regimens, a variety ofwhich will be readily apparent to one of skill in the art.

B. Ad-Mediated Delivery of Immunogenic Transgenes

The recombinant SAdV-39, SAdV-25.2, -26, -30, -37 or -38 vectors mayalso be employed as immunogenic compositions. As used herein, animmunogenic composition is a composition to which a humoral (e.g.,antibody) or cellular (e.g., a cytotoxic T cell) response is mounted toa transgene product delivered by the immunogenic composition followingdelivery to a mammal, and preferably a primate. A recombinant simian Adcan contain in any of its adenovirus sequence deletions a gene encodinga desired immunogen. The simian adenovirus is likely to be better suitedfor use as a live recombinant virus vaccine in different animal speciescompared to an adenovirus of human origin, but is not limited to such ause. The recombinant adenoviruses can be used as prophylactic ortherapeutic vaccines against any pathogen for which the antigen(s)crucial for induction of an immune response and able to limit the spreadof the pathogen has been identified and for which the cDNA is available.

Such vaccinal (or other immunogenic) compositions are formulated in asuitable delivery vehicle, as described above. Generally, doses for theimmunogenic compositions are in the range defined above for therapeuticcompositions. The levels of immunity of the selected gene can bemonitored to determine the need, if any, for boosters. Following anassessment of antibody titers in the serum, optional boosterimmunizations may be desired.

Optionally, a vaccinal composition of the invention may be formulated tocontain other components, including, e.g., adjuvants, stabilizers, pHadjusters, preservatives and the like. Such components are well known tothose of skill in the vaccine art. Examples of suitable adjuvantsinclude, without limitation, liposomes, alum, monophosphoryl lipid A,and any biologically active factor, such as cytokine, an interleukin, achemokine, a ligands, and optimally combinations thereof. Certain ofthese biologically active factors can be expressed in vivo, e.g., via aplasmid or viral vector. For example, such an adjuvant can beadministered with a priming DNA vaccine encoding an antigen to enhancethe antigen-specific immune response compared with the immune responsegenerated upon priming with a DNA vaccine encoding the antigen only.

The recombinant adenoviruses are administered in a “an immunogenicamount”, that is, an amount of recombinant adenovirus that is effectivein a route of administration to transfect the desired cells and providesufficient levels of expression of the selected gene to induce an immuneresponse. Where protective immunity is provided, the recombinantadenoviruses are considered to be vaccine compositions useful inpreventing infection and/or recurrent disease.

Alternatively, or in addition, the vectors of the invention may containa transgene encoding a peptide, polypeptide or protein which induces animmune response to a selected immunogen. The recombinant SAdV vectorsdescribed herein are expected to be highly efficacious at inducingcytolytic T cells and antibodies to the inserted heterologous antigenicprotein expressed by the vector.

For example, immunogens may be selected from a variety of viralfamilies. Example of viral families against which an immune responsewould be desirable include, the picornavirus family, which includes thegenera rhinoviruses, which are responsible for about 50% of cases of thecommon cold; the genera enteroviruses, which include polioviruses,coxsackieviruses, echoviruses, and human enteroviruses such as hepatitisA virus; and the genera apthoviruses, which are responsible for foot andmouth diseases, primarily in non-human animals. Within the picornavirusfamily of viruses, target antigens include the VP1, VP2, VP3, VP4, andVPG. Another viral family includes the calcivirus family, whichencompasses the Norwalk group of viruses, which are an importantcausative agent of epidemic gastroenteritis. Still another viral familydesirable for use in targeting antigens for inducing immune responses inhumans and non-human animals is the togavirus family, which includes thegenera alphavirus, which include Sindbis viruses, RossRiver virus, andVenezuelan, Eastern & Western Equine encephalitis, and rubivirus,including Rubella virus. The flaviviridae family includes dengue, yellowfever, Japanese encephalitis, St. Louis encephalitis and tick borneencephalitis viruses. Other target antigens may be generated from theHepatitis C or the coronavirus family, which includes a number ofnon-human viruses such as infectious bronchitis virus (poultry), porcinetransmissible gastroenteric virus (pig), porcine hemagglutinatingencephalomyelitis virus (pig), feline infectious peritonitis virus(cats), feline enteric coronavirus (cat), canine coronavirus (dog), andhuman respiratory coronaviruses, which may cause the common cold and/ornon-A, B or C hepatitis. Within the coronavirus family, target antigensinclude the E1 (also called M or matrix protein), E2 (also called S orSpike protein), E3 (also called HE or hemagglutin-elterose) glycoprotein(not present in all coronaviruses), or N (nucleocapsid). Still otherantigens may be targeted against the rhabdovirus family, which includesthe genera vesiculovirus (e.g., Vesicular Stomatitis Virus), and thegeneral lyssavirus (e.g., rabies).

Within the rhabdovirus family, suitable antigens may be derived from theG protein or the N protein. The family filoviridae, which includeshemorrhagic fever viruses such as Marburg and Ebola virus, may be asuitable source of antigens. The paramyxovirus family includesparainfluenza Virus Type 1, parainfluenza Virus Type 3, bovineparainfluenza Virus Type 3, rubulavirus (mumps virus), parainfluenzaVirus Type 2, parainfluenza virus Type 4, Newcastle disease virus(chickens), rinderpest, morbillivirus, which includes measles and caninedistemper, and pneumovirus, which includes respiratory syncytial virus.The influenza virus is classified within the family orthomyxovirus andis a suitable source of antigen (e.g., the HA protein, the N1 protein).The bunyavirus family includes the genera bunyavirus (Californiaencephalitis, La Crosse), phlebovirus (Rift Valley Fever), hantavirus(puremala is a hemahagin fever virus), nairovirus (Nairobi sheepdisease) and various unassigned bungaviruses. The arenavirus familyprovides a source of antigens against LCM and Lassa fever virus. Thereovirus family includes the genera reovirus, rotavirus (which causesacute gastroenteritis in children), orbiviruses, and cultivirus(Colorado Tick fever, Lebombo (humans), equine encephalosis, bluetongue).

The retrovirus family includes the sub-family oncorivirinal whichencompasses such human and veterinary diseases as feline leukemia virus,HTLVI and HTLVII, lentivirinal (which includes human immunodeficiencyvirus (HIV), simian immunodeficiency virus (SIV), felineimmunodeficiency virus (FIV), equine infectious anemia virus, andspumavirinal). Among the lentiviruses, many suitable antigens have beendescribed and can readily be selected. Examples of suitable HIV and SIVantigens include, without limitation the gag, pol, Vif, Vpx, VPR, Env,Tat, Nef, and Rev proteins, as well as various fragments thereof. Forexample, suitable fragments of the Env protein may include any of itssubunits such as the gp120, gp160, gp41, or smaller fragments thereof,e.g., of at least about 8 amino acids in length. Similarly, fragments ofthe tat protein may be selected. [See, U.S. Pat. No. 5,891,994 and U.S.Pat. No. 6,193,981.] See, also, the HIV and SIV proteins described in D.H. Barouch et al, J. Virol., 75(5):2462-2467 (March 2001), and R. R.Amara, et al, Science, 292:69-74 (6 Apr. 2001). In another example, theHIV and/or SIV immunogenic proteins or peptides may be used to formfusion proteins or other immunogenic molecules. See, e.g., the HIV-1 Tatand/or Nef fusion proteins and immunization regimens described in WO01/54719, published Aug. 2, 2001, and WO 99/16884, published Apr. 8,1999. The invention is not limited to the HIV and/or SIV immunogenicproteins or peptides described herein. In addition, a variety ofmodifications to these proteins has been described or could readily bemade by one of skill in the art. See, e.g., the modified gag proteinthat is described in U.S. Pat. No. 5,972,596. Further, any desired HIVand/or SIV immunogens may be delivered alone or in combination. Suchcombinations may include expression from a single vector or frommultiple vectors. Optionally, another combination may involve deliveryof one or more expressed immunogens with delivery of one or more of theimmunogens in protein form. Such combinations are discussed in moredetail below.

The papovavirus family includes the sub-family polyomaviruses (BKU andJCU viruses) and the sub-family papillomavirus (associated with cancersor malignant progression of papilloma). The adenovirus family includesviruses (EX, AD7, ARD, O.B.) which cause respiratory disease and/orenteritis. The parvovirus family feline parvovirus (feline enteritis),feline panleucopeniavirus, canine parvovirus, and porcine parvovirus.The herpesvirus family includes the sub-family alphaherpesvirinae, whichencompasses the genera simplexvirus (HSVI, HSVII), varicellovirus(pseudorabies, varicella zoster) and the sub-family betaherpesvirinae,which includes the genera cytomegalovirus (HCMV, muromegalovirus) andthe sub-family gammaherpesvirinae, which includes the generalymphocryptovirus, EBV (Burkitts lymphoma), infectious rhinotracheitis,Marek's disease virus, and rhadinovirus. The poxvirus family includesthe sub-family chordopoxvirinae, which encompasses the generaorthopoxvirus (Variola (Smallpox) and Vaccinia (Cowpox)), parapoxvirus,avipoxvirus, capripoxvirus, leporipoxvirus, suipoxvirus, and thesub-family entomopoxvirinae. The hepadnavirus family includes theHepatitis B virus. One unclassified virus which may be suitable sourceof antigens is the Hepatitis delta virus. Still other viral sources mayinclude avian infectious bursal disease virus and porcine respiratoryand reproductive syndrome virus. The alphavirus family includes equinearteritis virus and various Encephalitis viruses.

Immunogens which are useful to immunize a human or non-human animalagainst other pathogens include, e.g., bacteria, fungi, parasiticmicroorganisms or multicellular parasites which infect human andnon-human vertebrates, or from a cancer cell or tumor cell. Examples ofbacterial pathogens include pathogenic gram-positive cocci includepneumococci; staphylococci; and streptococci. Pathogenic gram-negativecocci include meningococcus; gonococcus. Pathogenic entericgram-negative bacilli include enterobacteriaceae; pseudomonas,acinetobacteria and eikenella; melioidosis; salmonella; shigella;haemophilus; moraxella; H. ducreyi (which causes chancroid); brucella;Franisella tularensis (which causes tularemia); yersinia (pasteurella);streptobacillus moniliformis and spirillum; Gram-positive bacilliinclude listeria monocytogenes; erysipelothrix rhusiopathiae;Corynebacterium diphtheria (diphtheria); cholera; B. anthracis(anthrax); donovanosis (granuloma inguinale); and bartonellosis.Diseases caused by pathogenic anaerobic bacteria include tetanus;botulism; other clostridia; tuberculosis; leprosy; and othermycobacteria. Pathogenic spirochetal diseases include syphilis;treponematoses: yaws, pinta and endemic syphilis; and leptospirosis.Other infections caused by higher pathogen bacteria and pathogenic fungiinclude actinomycosis; nocardiosis; cryptococcosis, blastomycosis,histoplasmosis and coccidioidomycosis; candidiasis, aspergillosis, andmucormycosis; sporotrichosis; paracoccidiodomycosis, petriellidiosis,torulopsosis, mycetoma and chromomycosis; and dermatophytosis.Rickettsial infections include Typhus fever, Rocky Mountain spottedfever, Q fever, and Rickettsialpox. Examples of mycoplasma andchlamydial infections include: mycoplasma pneumoniae; lymphogranulomavenereum; psittacosis; and perinatal chlamydial infections. Pathogeniceukaryotes encompass pathogenic protozoa and helminthes and infectionsproduced thereby include: amebiasis; malaria; leishmaniasis;trypanosomiasis; toxoplasmosis; Pneumocystis carinii; Trichans;Toxoplasma gondii; babesiosis; giardiasis; trichinosis; filariasis;schistosomiasis; nematodes; trematodes or flukes; and cestode (tapeworm)infections.

Many of these organisms and/or toxins produced thereby have beenidentified by the Centers for Disease Control [(CDC), Department ofHeath and Human Services, USA], as agents which have potential for usein biological attacks. For example, some of these biological agents,include, Bacillus anthracis (anthrax), Clostridium botulinum and itstoxin (botulism), Yersinia pestis (plague), variola major (smallpox),Francisella tularensis (tularemia), and viral hemorrhagic fevers[filoviruses (e.g., Ebola, Marburg], and arenaviruses [e.g., Lassa,Machupo]), all of which are currently classified as Category A agents;Coxiella burnetti (Q fever); Brucella species (brucellosis),Burkholderia mallei (glanders), Burkholderia pseudomallei (meloidosis),Ricinus communis and its toxin (ricin toxin), Clostridium perfringensand its toxin (epsilon toxin), Staphylococcus species and their toxins(enterotoxin B), Chlamydia psittaci (psittacosis), water safety threats(e.g., Vibrio cholerae, Crytosporidium parvum), Typhus fever (Richettsiapowazekii), and viral encephalitis (alphaviruses, e.g., Venezuelanequine encephalitis; eastern equine encephalitis; western equineencephalitis); all of which are currently classified as Category Bagents; and Nipan virus and hantaviruses, which are currently classifiedas Category C agents. In addition, other organisms, which are soclassified or differently classified, may be identified and/or used forsuch a purpose in the future. It will be readily understood that theviral vectors and other constructs described herein are useful todeliver antigens from these organisms, viruses, their toxins or otherby-products, which will prevent and/or treat infection or other adversereactions with these biological agents.

Administration of the SAdV-39, SAdV-25.2, -26, -30, -37 or -38 vectorsto deliver immunogens against the variable region of the T cells areanticipated to elicit an immune response including CTLs to eliminatethose T cells. In RA, several specific variable regions of TCRs whichare involved in the disease have been characterized. These TCRs includeV-3, V-14, V-17 and Vα-17. Thus, delivery of a nucleic acid sequencethat encodes at least one of these polypeptides will elicit an immuneresponse that will target T cells involved in RA. In MS, severalspecific variable regions of TCRs which are involved in the disease havebeen characterized. These TCRs include V-7 and Vα-10. Thus, delivery ofa nucleic acid sequence that encodes at least one of these polypeptideswill elicit an immune response that will target T cells involved in MS.In scleroderma, several specific variable regions of TCRs which areinvolved in the disease have been characterized. These TCRs include V-6,V-8, V-14 and Vα-16, Vα-3C, Vα-7, Vα-14, Vα-15, Vα-16, Vα-28 and Vα-12.Thus, delivery of a recombinant simian adenovirus that encodes at leastone of these polypeptides will elicit an immune response that willtarget T cells involved in scleroderma.

C. Ad-Mediated Delivery Methods

The therapeutic levels, or levels of immunity, of the selected gene canbe monitored to determine the need, if any, for boosters. Following anassessment of CD8+T cell response, or optionally, antibody titers, inthe serum, optional booster immunizations may be desired. Optionally,the recombinant SAdV-39, SAdV-25.2, -26, -30, -37 or -38 vectors may bedelivered in a single administration or in various combination regimens,e.g., in combination with a regimen or course of treatment involvingother active ingredients or in a prime-boost regimen. A variety of suchregimens has been described in the art and may be readily selected.

For example, prime-boost regimens may involve the administration of aDNA (e.g., plasmid) based vector to prime the immune system to second,booster, administration with a traditional antigen, such as a protein ora recombinant virus carrying the sequences encoding such an antigen.See, e.g., WO 00/11140, published Mar. 2, 2000, incorporated byreference. Alternatively, an immunization regimen may involve theadministration of a recombinant SAdV-39, SAdV-25.2, -30, -37 or -38vector to boost the immune response to a vector (either viral orDNA-based) carrying an antigen, or a protein. In still anotheralternative, an immunization regimen involves administration of aprotein followed by booster with a vector encoding the antigen.

In one embodiment, a method of priming and boosting an immune responseto a selected antigen by delivering a plasmid DNA vector carrying saidantigen, followed by boosting with a recombinant SAdV-39, SAdV-25.2,-26, -30, -37 or -38 vector is described. In one embodiment, theprime-boost regimen involves the expression of multiproteins from theprime and/or the boost vehicle. See, e.g., R. R. Amara, Science,292:69-74 (6 Apr. 2001) which describes a multiprotein regimen forexpression of protein subunits useful for generating an immune responseagainst HIV and SIV. For example, a DNA prime may deliver the Gag, Pol,Vif, VPX and Vpr and Env, Tat, and Rev from a single transcript.Alternatively, the SIV Gag, Pol and HIV-1 Env is delivered in arecombinant SAdV-39, SAdV-25.2, -26, -30, -37 or -38 adenovirusconstruct. Still other regimens are described in WO 99/16884 and WO01/54719.

However, the prime-boost regimens are not limited to immunization forHIV or to delivery of these antigens. For example, priming may involvedelivering with a first SAdV-39, SAdV-25.2, -26, -30, -37 or -38 vectorfollowed by boosting with a second Ad vector, or with a compositioncontaining the antigen itself in protein form. In one example, theprime-boost regimen can provide a protective immune response to thevirus, bacteria or other organism from which the antigen is derived. Inanother embodiment, the prime-boost regimen provides a therapeuticeffect that can be measured using convention assays for detection of thepresence of the condition for which therapy is being administered.

The priming composition may be administered at various sites in the bodyin a dose dependent manner, which depends on the antigen to which thedesired immune response is being targeted. The amount or situs ofinjection(s) or to pharmaceutical carrier is not a limitation. Rather,the regimen may involve a priming and/or boosting step, each of whichmay include a single dose or dosage that is administered hourly, daily,weekly or monthly, or yearly. As an example, the mammals may receive oneor two doses containing between about 10 μg to about 50 μg of plasmid incarrier. A desirable amount of a DNA composition ranges between about 1μg to about 10,000 μg of the DNA vector. Dosages may vary from about 1μg to 1000 μg DNA per kg of subject body weight. The amount or site ofdelivery is desirably selected based upon the identity and condition ofthe mammal.

The dosage unit of the vector suitable for delivery of the antigen tothe mammal is described herein. The vector is prepared foradministration by being suspended or dissolved in a pharmaceutically orphysiologically acceptable carrier such as isotonic saline; isotonicsalts solution or other formulations that will be apparent to thoseskilled in such administration. The appropriate carrier will be evidentto those skilled in the art and will depend in large part upon the routeof administration. The compositions described herein may be administeredto a mammal according to the routes described above, in a sustainedrelease formulation using a biodegradable biocompatible polymer, or byon-site delivery using micelles, gels and liposomes. Optionally, thepriming step also includes administering with the priming composition, asuitable amount of an adjuvant, such as are defined herein.

Preferably, a boosting composition is administered about 2 to about 27weeks after administering the priming composition to the mammaliansubject. The administration of the boosting composition is accomplishedusing an effective amount of a boosting composition containing orcapable of delivering the same antigen as administered by the primingDNA vaccine. The boosting composition may be composed of a recombinantviral vector derived from the same viral source (e.g., adenoviralsequences of the invention) or from another source. Alternatively, the“boosting composition” can be a composition containing the same antigenas encoded in the priming DNA vaccine, but in the form of a protein orpeptide, which composition induces an immune response in the host. Inanother embodiment, the boosting composition contains a DNA sequenceencoding the antigen under the control of a regulatory sequencedirecting its expression in a mammalian cell, e.g., vectors such aswell-known bacterial or viral vectors. The primary requirements of theboosting composition are that the antigen of the composition is the sameantigen, or a cross-reactive antigen, as that encoded by the primingcomposition.

In another embodiment, the SAdV-39, SAdV-25.2, -26, -30, -37 or -38vectors are also well suited for use in a variety of other immunizationand therapeutic regimens. Such regimens may involve delivery of SAdV-39,SAdV-25.2, -26, -30, -37 or -38 vectors simultaneously or sequentiallywith Ad vectors of different serotype capsids, regimens in whichSAdV-39, SAdV-25.2, -26, -30, -37 or -38 vectors are deliveredsimultaneously or sequentially with non-Ad vectors, regimens in whichthe SAdV-39, SAdV-25.2, -30, -37 or -38 vectors are deliveredsimultaneously or sequentially with proteins, peptides, and/or otherbiologically useful therapeutic or immunogenic compounds. Such uses willbe readily apparent to one of skill in the art.

In still another embodiment, the invention provides the use of capsid ofthese viruses (optionally an intact or recombinant viral particle or anempty capsid) is used to induce an immunomodulatory effect response, orto enhance or adjuvant a cytotoxic T cell response to another activeagent by delivering an adenovirus SAdV-39, SAdV-25.2, -26, -30, -37 or-38 to subject. The SAdV-39, SAdV-25.2, -26, -30, -37 or -38 capsid canbe delivered alone or in a combination regimen with an active agent toenhance the immune response thereto. Advantageously, the desired effectcan be accomplished without infecting the host with a subgroup Eadenovirus. In another aspect, a method of inducing interferon alphaproduction in a subject in need thereof comprising delivering theSAdV-39, SAdV-25.2, -26, -30, -37 or -38 capsid to a subject isprovided. In still another aspect, a method for producing one or morecytokines (e.g., IFN-α)/chemokines in culture is provided. This methodinvolves incubating a culture containing dendritic cells and theSAdV-39, SAdV-25.2, -26, -30, -37 or -38 capsid described herein underconditions suitable to produce cytokines/chemokines, including, alphainterferon, among others.

The cytokines so produced are useful in a variety of applications. Forexample, in the case of IFNα, the production described herein isparticularly desirable, as it is believed that it will provideadvantages over commercially available recombinantly produced IFNα,which contain only one or two subtypes of IFNα produced in bacteria. Incontrast, the method is anticipated to produce multiple subtypes ofnatural human IFNα, which is expected to result in a broader spectrum ofaction. It is believed that each subtype employs a specific biologicalactivity. Further, it is anticipated that the natural interferonproduced by the method provided herein will be immunologicallyindistinguishable from the patient's naturally produced interferon,thereby reducing the risk of the drug being rejected by the subject'simmune system, usually caused by the formation of neutralizingantibodies against recombinantly produced interferons.

The following examples illustrate the cloning of SAdV-39, SAdV-25.2,-26, -30, -37 or -38 and the construction of exemplary recombinantSAdV-39, SAdV-25.2, -26, -30, -37 or -38 vectors. These examples areillustrative only, and do not limit the scope of the present invention.

Example 1 Isolation of Simian Adenoviruses

Stool samples were obtained from the chimpanzee colony at the Universityof Louisiana New Iberia Research Center, 4401 W. Admiral Doyle Drive,New Iberia, La., USA, and from the chimpanzee colony at the Michael E.Keeling Center for Comparative Medicine and Research, University ofTexas M. D. Anderson Cancer Center, Bastrop, Tex., USA. Supernatantsfrom the chimpanzee stool samples in suspension in Hanks' Balanced Saltsolution were sterile filtered through 0.2 micron syringe filters. 100μl of each filtered sample was inoculated into the human cell line A549cultures. These cells were grown in Ham's F12 with 10% FBS, 1%Penn-Strep and 50 μg/ml gentamicin. After about 1 to 2 weeks in culture,visual cytopathic effect (CPE) was obvious in cell cultures with severalof the inocula. The adenoviruses were purified from cultures in A549cells using standard published cesium chloride gradient techniques foradenovirus purification. DNA from the purified adenoviruses was isolatedand completely sequenced by Qiagen Genomic services, Hilden, Germany.

Based on the phylogenetic analysis of the viral DNA sequences, theadenoviruses designated simian adenovirus 25.2 (SAdV-25.2), simianadenovirus 26 (SAdV-26), simian adenovirus 30 (SAdV-30), simianadenovirus 37 (SAdV-37), simian adenovirus 38, (SAdV-38) and simianadenovirus 39 (SAdV-39) were determined to be in the same subgroup ashuman subgroup E.

Sequence analysis revealed that the closest hexon match to the hexon ofthe SAdV-26 is chimpanzee adenovirus 6 (98.4 percent (%) identity) andthe closest fiber match is human adenovirus 4 (93% identity).

Sequence analysis revealed that the closest genomic match of theSAdV25.2 virus is simian (chimpanzee) adenovirus 25 [Genbank accessionnumber AC000011]. SAdV25 was previously named C68 or Pan9 [U.S. Pat. No.6,083,716]. At the nucleic acid level, there is 94% identity betweenSAdV25.2 and SAdV25 as determined by vector NTI-AlignX. At the hexon(amino acid) level, SAdV-25.2 has 99% identity to simian adenovirus 25with two conservative amino acid changes and two non-conservativechanges. The following table shows the amino acid changes in SAdV25.2 ascompared to the SAdV25 hexon sequence, with reference to the hexon ofSAdV25.2 provided herewith in SEQ ID NO: 140. The numbering of bothsequences is identical.

Amino Acid Residue SEQ ID NO: 140 Change SAdV-25.2 (vs. SAdV25) 181 Glu(Lys) (non-conservative) 404 Lys (Arg) 477 Ala (Thr) (non-conservative)497 Ala (Ser) 838 (Ala) Thr (non-conservative)

The methodology used to create the vectors was to first create abacterial plasmid molecular clone of the entire E1-deleted adenoviralvector followed by transfection of the plasmid DNA into the E1complementing cell line HEK 293 to rescue the viral vector.

In order to create molecular clones of an E1-deleted adenoviral vector,plasmid molecular clones of the E1-deleted adenoviruses were firstcreated where recognition sites for the rare-cutting restriction enzymesI-CeuI and PI-SceI have been inserted in place of an E1 deletion.Expression cassettes flanked by I-CeuI and PI-SceI, and excised usingthese restriction enzymes, were ligated into the E1-deleted adenoviralplasmid clones. The plasmid adenoviral molecular clone harboring thedesired expression cassette in place of the E1 deletion were transfectedinto HEK 293 cells to rescue the recombinant adenoviral vectors. Rescuefollowing transfection was found to be facilitated by first releasingthe linear adenoviral genome from the plasmid by restriction enzymedigestion.

Example 2 Construction of an E1-Deleted Plasmid Molecular Clones Basedon SAdV-39, SAdV-25.2, SAdV-26, SAdV-30, SAdV-37, or SAdV-38 UsingStandard Molecular Biology Techniques

A. Vector Construction of SAdV-39

An E1 deleted vector using the SAdV-39 (subgroup E) was prepared asdescribed.

1. Construction of pSR3:

A linker containing SmaI, HindII, EcoRV sites flanked by SwaI sites wascloned into pBR322 cut with EcoRI and NdeI as follows.

The oligomers SEQ ID NO: 196: SV25 Top:AATTATTTAAATCCCGGGTATCAA-GCTTGATAGATATCATTTAAAT and SEQ ID NO: 197: SV25Bot TAATTTAAATGATATCTATCAAGCTTGATACCCGGGATTTAAAT were annealed togetherto create the linker.

2. Cloning of the SAdV-39 Viral Left End to the HindIII site (7152)

The viral DNA was digested with HindIII and the 7152 bp left endfragment was cloned into pSR3 digested with SmaI and HindIII to yieldpSR3 C39 LE.

3. E1 Functional Deletion and Insertion of I-CeuI and PI-SceI Sites:

The plasmid pC39LE was deleted between SnaBI and NdeI (Klenow filled in)to delete E1 and most of E1b coding regions; in its place a DNA fragment(the EcoRV fragment from pBleuSK I-PI harboring sites for I-CeuI andPI-SceI) was ligated in to yield pC39LEIP.

4. Cloning of the SAdV-39 Viral Right End from the NheI Site (35779).

The SAdV-39 viral DNA was digested with NheI and the 775 by right endfragment was cloned into pC39LEIP between EcoRV and NheI to yield pC39LEIP RE.

5. Cloning of the SAdV-39 Viral NheI (3033-35779) Fragment

The plasmid pC37-LE-IP-RE was digested with HindIII and the 32746 bpviral NheI fragment was ligated in. The clone with the correctorientation was called pC39 IP.

B. Construction of an E1-Deleted Plasmid Molecular Clone Based onSAdV-25.2, Using Standard Molecular Biology Techniques

An E1 deleted vector using the SAdV-25.2 (subgroup E) was prepared asdescribed.

1 Construction of pSR6:

A linker containing SmaI, AscI, AvrII, EcoRV sites flanked by PacI sitesis cloned into pBR322 cut with EcoRI and NdeI as follows.

The oligomers SEQ ID NO: 198: pSR6 top:AATTTTAATTAACCCGGGTATCGGC-GCGCCTTAACCTAGGGATAGATATCTTAATTAA and SEQ IDNO: 199: pSR6 bot:TATTAATTAAGATATCTATCCCTAGGTTAAGGCGCGCCGATACCCGGGTTAA-TTAA were annealedtogether to create the linker.

2. Cloning of the Viral Left End to the AscI Site (7959)

The viral DNA was digested with AscI and the 7959 bp left end fragmentwas cloned into pSR6 digested with SmaI and AscI to yield pSR5 C25.2 LE

3. E1 functional deletion and insertion of I-CeuI and PI-SceI sites:

The plasmid pSR5 C25.2 LE was digested with SnaBI+NdeI; the NdeI sitewas filled in with Klenow. The EcoRV fragment from pBleuSK I-PI wasligated in to create pSR5 C25.2 LE IP.

4. Cloning of the Viral Right End from the XbaI Site (30071):

The plasmid pSR5 C25.2 LE IP was digested with XbaI+EcoRV. The 6559 bpright end (XbaI digest) fragment from the SAdV-25.2 DNA was ligated into create pAdC12-LE-IP-RE.

5. Cloning of the Viral Middle XbaI Fragment (6037-30071)

The plasmid pAdC12-LE-IP-RE was digested with XbaI. The 24034 byfragment from the SAdV-25.2 DNA was ligated in to create pAdC25.2 IP.

C. Construction of an E1-Deleted Plasmid Molecular Clone Based onSAdV-26, Using Standard Molecular Biology Techniques

An E1 deleted vector using the SAdV-26 (subgroup E) was prepared asdescribed.

1. Construction of pSR5:

A linker containing SmaI, ClaI, XbaI, SpeI, EcoRV sites flanked by SwaIis cloned into pBR322 cut with EcoRI and NdeI.

The synthetic oligonucleotides SV39T, SEQ ID NO: 194AATTATTTAAATCCCGGGGATCATCGATGATCTCTAGAGATCACTAGTCTAGGAT ATCATTTAAA andSV39B, SEQ ID NO: 195TATTTAAATGATATCCTAGACTAGT-GATCTCTAGAGATCATCGATGATCCCCGGGATTTAAAT wereannealed to create the linker.

2. Cloning of the Viral Left End to the XbaI Site (6029)

The viral DNA was digested with XbaI and the 6 kb fragments (left andright ends) were gel purified and ligated into pSR5 digested with SmaIand XbaI.

3. E1 Functional Deletion and Insertion of I-CeuI and PI-SceI Sites:

The plasmid pSR5-C12-LE was digested with SnaBI+NdeI; the NdeI site wasfilled in with Klenow. The EcoRV fragment from pBleuSK I-PI was ligatedin to create pAdC12-LE-IP.

4. Cloning of the Viral Right End from the XbaI Site (30158):

The plasmid pAdC12-LE-IP was digested with XbaI+EcoRV. The 6471 bp rightend (XbaI digest) fragment from the SAdV-26 DNA was ligated in to createpAdC12-LE-IP-RE.

5. Cloning of the Viral Middle XbaI Fragment (6029-30158)

The plasmid pAdC12-LE-IP-RE was digested with XbaI+EcoRV. The 24129 bpfragment from the SAdV-26 DNA was ligated in to create pC26 IP.

D. Vector Construction of SAdV-30

An E1 deleted vector using the SAdV-30 (subgroup E) was prepared asdescribed.

1. Construction of pSR3:

A linker containing SmaI, HindII EcoRV sites flanked by SwaI sites wascloned into pBR322 cut with EcoRI and NdeI as follows.

The oligomers SEQ ID NO: 196: SV25 Top:AATTATTTAAATCCCGGGTATCAAGCTTGATAGATATCATTTAAAT and SEQ ID NO: 197: SV25Bot: TAATTTAAATGATATCTATCAAGCTTGATACCCGG-GATTTAAAT were annealedtogether to create the linker.

2. Cloning of the Viral Left End to the HindIII Site (7146)

The viral DNA was digested with HindIII and the 7146 bp left endfragment was cloned into pSR3 digested with SmaI and HindIII to yieldpSR3 C30 LE

3. E1 Functional Deletion and Insertion of I-CeuI and PI-SceI Sites:

The plasmid pSR3C30 LE was digested with SnaBI+NdeI; the NdeI site wasfilled in with Klenow. The EcoRV fragment from pBleuSK I-PI was ligatedin to create pC30 LE IP. The internal EcoRI site (at position 1040 bpfrom the beginning of the left ITR) was destroyed by digesting pC30 LEIP with EcoRI, filling in the overhangs with Klenow polymerase andre-ligating. This yielded the plasmid pC30 LE IP (EcoRI del).

4. Cloning of the Viral Right End from the HindIII Site (33048):

The plasmid pC30 LE IP (EcoRI del) was digested with HindIII+EcoRV. The3574 bp right end (HindIII digest) fragment from the SAdV-30 DNA wasligated in to create pC30-LE-IP-RE.

5. Cloning of the Viral Middle XbaI (6035) to EcoRI (33631) Fragment

The plasmid pC30-LE-IP-RE was digested with XbaI+HindIII. The 27596 bpfragment from the SAdV-30 DNA was ligated in to create pC30 IP.

E. Vector Construction of SAdV-37

An E1 deleted vector using the SAdV-37 (subgroup E) was prepared asdescribed.

1. Construction of pSR3:

A linker containing SmaI, HindII, EcoRV sites flanked by SwaI sites wascloned into pBR322 cut with EcoRI and NdeI as follows. The oligomers:SEQ ID NO: 196: SV25 Top:AATTATTTAAATCCCGGGTATCAAGCTTGATAGAT-ATCATTTAAAT and SEQ ID NO: 197: SV25Bot: TAATTTAAATGATATCTATCAAGCTTGATACCCGGGATTTAAAT were annealed togetherto create the linker.

2. Cloning of the SAdV-37 Viral Left End to the HindIII Site (7147)

The viral DNA was digested with HindIII and the 7147 bp left endfragment was cloned into pSR3 digested with SmaI and HindIII to yieldpSR3 C37 LE

3. E1 Functional Deletion and Insertion of I-CeuI and PI-SceI Sites:

The plasmid pSR3 C37LE was deleted between SnaBI and NdeI (Klenow filledin) to delete E1a and most of E1b coding regions; in its place a DNAfragment (the EcoRV fragment from pBleuSK I-PI harboring sites forI-CeuI and PI-SceI) was ligated in to yield pSR3 C37 LE IP.

4. Cloning of the SAdV-37 Viral Right End from the HindIII Site (33048).

The plasmid pC37 LE IP was digested with HindIII+EcoRV. The 3575 bpright end (HindIII digest) fragment from the SAdV-37 DNA was ligated into create pC37-LE-IP-RE.

5. Cloning of the SAdV-37 Viral HindIII (23522-33060) Fragment

The plasmid pC37-LE-IP-RE was digested with HindIII and the 9538 bpviral HindIII fragment was ligated in. The clone with the correctorientation was called pC37 del Xba Pac.

6. Cloning of the SAdV-37 Viral XbaI (6036) PacI (30181) Fragment

The plasmid pC37 del Xba Pac was digested with XbaI and PacI the 24145bp viral XbaI-PacI fragment was ligated in to yield pC37IP.

F. Construction of E1-Deleted Adenoviral Vectors

In order to insert a DNA segment harboring I-CeuI and PI-SceIrecognition sites in place of an E1 deletion, the plasmid pBleuSK I-PIwas used. The plasmid pBleuSK I-PI contains a 654 bp fragment insertedinto the EcoRV site of pBluescript II SK(+) (Stratagene). The 654 bpsegment harbors recognition sites for the rare-cutter restrictionenzymes I-CeuI and PI-SceI. In order to insert a DNA segment harboringI-CeuI and PI-SceI recognition sites in place of an E1 deletion, pBleuSKI-PI was digested with EcoRV and the 654 bp fragment was ligated intothe location of the adenoviral genome E1 deletion. The sequence of theinserted DNA is shown below flanked by EcoRV recognition sites. Therecognition sequences for I-CeuI and PI-SceI are underlined.

SEQ ID NO: 200: GATATCATTTCCCCGAAAAGTGCCACCTGACGTAACTATAACGGTCCTAAGGTAGCGAAAGCTCAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGGTACGAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCAGATCTGCAGATCTGAATTCATCTATGTCGGGTGCGGAGAAAGAGGTAATGAAATGGCATTATGGGTATTATGGGTCTGCATTAATGAATCG GCCAGATATC

In order to construct E1-deleted adenoviral vectors expressing theinfluenza virus nucleoprotein, the nucleotide sequence encoding the H1N1influenza A virus NP (A/Puerto Rico/8/34/Mount Sinai, GenBank accessionnumber AF389119.1) was codon optimized and completely synthesized(Celtek Genes, Nashville, Tenn.). An expression cassette composed of thehuman cytomegalovirus early promoter, a synthetic intron (obtained fromthe plasmid pCI (Promega, Madison, Wis.), the codon optimized influenzaA NP coding sequence and the bovine growth hormone polyadenylationsignal was constructed. The plasmid pShuttle CMV PI FluA NP harbors theabove described expression cassette where it is flanked by therecognition sites for the rare-cutting restriction enzymes I-CeuI andPI-SceI (New England Biolabs) respectively. In order to create amolecular clone of an E1-deleted adenoviral vector, plasmid molecularclones of the E1-deleted adenoviruses were created as described in thepreceding portions of this example, where recognition sites for therare-cutting restriction enzymes I-CeuI and PI-SceI were inserted inplace of an E1 deletion. The E1-deleted adenoviral plasmids were thendigested with I-CeuI and PI-SceI and the expression cassette (digestedby the same enzymes) was ligated in. The resulting adenoviral plasmidmolecular clones were transfected into HEK 293 cells to rescuerecombinant adenoviral vector. Rescue following transfection was foundto be facilitated by first releasing the linear adenoviral genome fromthe plasmid by restriction enzyme digestion.

Example 3 Assessment of Cross-Neutralizing Antibodies

Wild-type SAdV-39, SAdV-25.2, SAdV-26, SAdV-30, SAdV-37 and SAdV-38 wereassessed for cross-neutralizing activity as compared to human Adenovirus5 (subspecies C) and chimpanzee adenovirus 7 (SAdV-24), and human pooledIgG using an infection inhibition neutralizing antibody assay monitoredby direct immunofluorescence. The human pooled IgG [Hu Pooled IgG] ispurchased commercially and is approved for administration inimmunocompromised patients, as it contains antibodies against a numberof antigens to which the general human population is exposed. Thepresence or absence of neutralizing antibodies to the simianadenoviruses for the human pooled IgG is a reflection of the prevalenceof antibodies to these adenoviruses in the general population.

The assay was performed as follow. Serum samples obtained from rabbitspreviously injected with HAdV-5 or SAdV-24 were heat inactivated at 56°C. for 35 min. Wild type adenovirus (10⁸ particles/well) was diluted inserum-free Dulbecco's modified Eagle's medium (DMEM) and incubated with2-fold serial dilutions of heat-inactivated serum samples in DMEM for 1h at 37° C. Subsequently, the serum-adenovirus mixture was added toslides in wells with 105 monolayer A549 cells. After 1 hr, the cells ineach well were supplemented with 100 μl of 20% fetal bovine serum(FBS)-DMEM and cultured for 22 h at 37° C. in 5% CO2. Next, cells wererinsed twice with PBS and stained with DAPI and a goat, FITC labeled,broadly cross reactive antibody (Virostat) raised against HAdV-5following fixation in paraformaldehyde (4%, 30 min) and permeabilizationin 0.2% Triton (4° C., 20 min). The level of infection was determined bycounting the number of FITC positive cells under microscopy. The NABtiter is reported as the highest serum dilution that inhibitedadenovirus infection by 50% or more, compared with the naive serumcontrol. Where a titer value of <1/20 is shown, the neutralizingantibody concentration was under the limit of detection, i.e., 1/20.

Anti- Species Virus Anti-HAdV-5 SAdV-24 H. Pooled IgG C HAdV-5   1/81,920 <1/20      1/640 E SAdV-24 (C7) <1/20 1/655,360 1/20 ESAdV-25.2 <1/20 1/655,360 1/40 SAdV-26  1/20 1/40,960  1/20 SAdV-30<1/20 1/1,280  1/20 SAdV-37 <1/20 1/320    1/20 SAdV-39 <1/20 1/320   1/20

These data indicate that there is minimal immunoreactivity to theseadenoviruses in the general population. These data further indicate thatthe simian adenoviruses in the preceding Table which do not cross-reactwith HAdV-5 and SAdV-24 may be used in regimens which involve sequentialdelivery of adenoviruses, e.g., prime-boost or cancer therapies.

Example 4 Cytokine Induction

Plasmacytoid dendritic cells were isolated from human peripheral bloodmononuclear cells (PBMCs) and cultured in medium in 96 well plates andinfected with adenoviruses. 48 hrs later the cells are spun down and thesupernatant collected and analyzed for the presence of interferon α.

More specifically, the PBMCs were obtained from the Center For AIDSResearch (CFAR) immunology core at the University of Pennsylvania. 300million of these cells were then used for isolating plasmacytoiddendritic cells (pDCs) using the “human plasmacytoid dendritic cellisolation kit” from Miltenyi Biotec as per the instructions providedalong with the kit. The isolation using this kit was based on removingall other cell types but pDCs from PBMCs.

The final cell numbers usually vary from donor to donor, but range from0.4-0.7 million cells. So the data that has been generated (discussedbelow) comes from analysis of cells from multiple donors. Surprisinglythough, the separation of subgroups based on interferon or othercytokine release is maintained even when analyzing cells from multipledonors.

The cells were cultured in RPMI-1640 medium (Mediatech) supplementedwith L-glutamine, 10% Fetal bovine serum (Mediatech), 10 mM Hepes buffersolution (Invitrogen), antibiotics (Penicillin, streptomycin andGentamicin—from Mediatech) and human-interleukin 3 (20 ng/mL —R&D).Wild-type adenoviruses were directly added to the cells at amultiplicity of infection (MOI) of 10,000 (10,000 viral particles percell, with a concentration of 10⁶ cells/ml). 48 hrs later the cells werespun down and the supernatant assayed for the presence of interferon.Cytokines were measured using an enzyme-linked immunosorbent assay(ELISA) kit from PBL Biomedical Laboratories using the recommendedprotocol from the manufacturer.

The study showed that subgroup C adenoviruses produced no detectableamounts of IFNα (the assay has a detection limit of 1250 pg/mL). Incontrast, all tested members of the subgroup E adenoviruses producedIFNα and, in general, produced significantly more IFNα as compared tothe subgroup B adenoviruses.

A variety of other cytokines were also detected in the screening of theadenoviruses. However, in general, the subgroup E adenoviruses producedsignificantly higher levels of IL-6, RANTES, MIP-1α, TNF-α, IL-8, andIP-10 than the subgroup C adenoviruses. The subgroup B adenoviruses alsooutperformed the subgroup C adenoviruses in induction of IFNα, IL-6,RANTES, and MIP1α.

Since no significant cell lysis was observed in this study, thissuggests that the cytokine is produced by contacting the cells with thesubgroup E adenovirus, without regard to infection and in the absence ofany significant amount of viral replication.

In another study (not shown), cells were incubated as described abovewith either empty C7 capsid proteins (Ad subgroup E) or UV-inactivatedadenovirus C7 viral vector (UV inactivation causes cross-linking,eliminating adenovirus gene expression). In these studies, the same orhigher levels of IFNα were observed for both the empty capsid and theinactivated viral vector as compared to intact C7.

The inventors have found that exposing cytokine-producing cells orchemokine-producing cells, such as PBMC, PBL, and dendritic cells, to acapsid from a member of the subgroup E adenovirus induces cytokines, andin particular, IFNα, or chemokines in amounts significantly higher thanare induced by other adenovirus subgroups. Thus, the members of thissubgroup are useful for inducing alpha interferon and, in smallerquantities, a number of other cytokines/chemokines in culture.

In the case of IFNα, the production method is particularly desirable, asit is believed to be advantageous over recombinantly produced IFNα. Incontrast, the method provided herein is anticipated to produce multiplesubtypes of natural human IFNα, which is expected to result in a broaderspectrum of action. It is believed that each subtype employs a specificbiological activity. Further, it is anticipated that the naturalinterferon produced by the method provided herein will beimmunologically indistinguishable from the patient's naturally producedinterferon, thereby reducing the risk of the drug being rejected by thepatient's immune system, usually caused by the formation of neutralizingantibodies against recombinantly produced interferons.

Other cytokines produced by the subgroup E adenoviruses include,interleukin (IL)-6, IL-8, IP-10, macrophage inflammatory protein -1alpha (MIP-1α), RANTES, and tumor necrosis factor alpha. Methods ofpurifying these cytokines/chemokines from culture and therapeutic oradjuvant uses of these cytokines/chemokines have been described in theliterature. Further, commercially available columns or kits may used forpurification of the cytokines/chemokines prepared according to theinvention. The cytokines/chemokines produced using the invention may beformulated for use in a variety of indications.

For example, cytokines described herein include, interferon alpha(IFNα), tumor necrosis factor alpha (TNFα), IP-10 (Interferon gammaInducible Protein), interleukin-6 (IL-6), and IL-8. IFNα, has beendescribed as being useful in treatment of influenza, hepatitis(including, e.g., hepatitis B and C), and a variety of neoplasms, e.g.,kidney (renal cell carcinoma), melanoma, malignant tumor, multiplemyeloma, carcinoid tumor, lymphoma and leukemia (e.g., chronicmyelogenous leukemia and hairy cell leukemia). A mixture of IFNαsubtypes produced as described herein can be purified using knowntechniques. See, e.g., WO 2006/085092, which describes the use ofmonoclonal antibodies and column purification. Other techniques havebeen described in the literature.

IFNα produced as described herein can be purified using known methods.See, e.g., U.S. Pat. No. 4,680,260, U.S. Pat. No. 4,732,683, and G.Allen, Biochem J., 207:397-408 (1982). TNFα has been described as beinguseful in treatment in autoimmune disorders including, e.g., psoriasisand rheumatoid arthritis. IP-10, Interferon gamma Inducible Protein, canbe used as a potent inhibitor of angiogenesis and to have a potentthymus-dependent anti-tumor effect.

Thus, in still another aspect, a method for producing IFNα by incubatinga culture containing dendritic cells and a subgroup E adenovirus capsidunder conditions suitable to produce cytokines is provided.

In one embodiment, blood is drawn from healthy donors (preferably human)and peripheral blood leukocytes (PBL) or peripheral blood mononuclearcells (PBMC) are prepared using known techniques. In one embodiment, PBLare used as the cytokine-producing cells according to the method of theinvention. In another embodiment, PBMC are used as thecytokine-producing cells. In another embodiment, plasmacytoid dendriticcells are isolated from the PBL or PBMC using known techniques, e.g.,using the commercially available kit “human plasmacytoid dendritic cellisolation kit” by Miltenyi Biotec GmbH (Germany). The selected cells arecultured in suspension with an appropriate media and the adenovirussubgroup E capsid protein. Appropriate media can be readily determinedby one of skill in the art. However, in one embodiment, the media is aRPMI-1640 medium. Alternatively, other media may be readily selected.

The cells may be cultured in a suitable vessel, e.g., a microtiter well,a flask, or a larger vessel. In one embodiment, the concentration of thecells is about 1 million cells/mL culture media. However, other suitablecell concentrations may be readily determined by one of skill in theart.

Advantageously, the invention does not require the use of interferons asprimers. However, if desired, the media may include a suitable cytokine,IL-3, in order to stimulate cell growth. One suitable concentration isabout 20 ng/mL. However, other concentrations may be used.

In one embodiment, the adenovirus capsid protein is introduced into theculture containing the cells. The adenovirus capsid protein can bedelivered to the culture in any of the forms described herein (e.g., aviral particle, including an empty capsid particle, a viral vectorhaving an Ad subgroupE capsid, and the like). Typically the capsidprotein will be suspended in a suitable carrier, e.g., culture media,saline, or the like.

Suitably, the adenovirus subgroup E capsids are added to the culture inan amount of about 100 to 100,000 adenovirus subgroup E particles percell. The mixture is then incubated, e.g., in the range of about 28° C.to about 40° C., in the range from about 35° C. to about 37° C., orabout 37° C.

Typically, approximately 12 to 96 hours, or about 48 hours later, cellsare spun down and the supernatant is collected. Suitably, this isperformed under conditions which avoid cell lysis, thereby reducing oreliminating the presence of cellular debris in the supernatant.Centrifugation permits separation of the cytokines from the cells,thereby providing a crudely isolated cytokine. Sizing columns, and otherknown columns and methods are available for further purification ofcytokines from adenoviruses and adenoviral capsids, and the like.

These cytokines, so purified, are available for formulation and use in avariety of applications.

As described herein and without being bound by theory, the immuneenhancing and/or cytokine producing ability of the adenovirus subgroup Eappears to be based on contact between the cells and the adenoviralcapsid, without regard to infectivity or replication ability of theadenoviral particle. Thus, in one embodiment, an empty adenovirussubgroup E particle (i.e., an adenoviral capsid having no DNA packagedtherein which expresses any adenoviral or transgene product) isdelivered to the cells. In another embodiment, a non-infectiouswild-type subgroup E particle or a recombinant adenoviral vectorpackaged in an adenoviral subgroup E capsid (particle) is used. Suitabletechniques for inactivating such viral particles are known in the artand may include without limitation, e.g., UV irradiation (whicheffectively cross-links genomic DNA preventing expression).

All documents recited above are incorporated herein by reference.Numerous modifications and variations are included in the scope of theabove-identified specification and are expected to be obvious to one ofskill in the art. Such modifications and alterations to the compositionsand processes, such as selections of different minigenes or selection ordosage of the vectors or immune modulators are believed to be within thescope of the claims appended hereto.

1. An adenovirus having a capsid comprising a capsid protein selectedfrom the group consisting of: (a) a hexon protein of SAdV-39, aminoacids 1 to 940 of SEQ ID NO: 11; a hexon protein of SAdV-30, amino acids1 to 938 of SEQ ID NO: 108; a hexon protein of SAdV-25.2, amino acids 1to 933 of SEQ ID NO: 140; a hexon protein of SAdV-37, amino acids 1 to942 of SEQ ID NO: 43; a hexon protein of SAdV-38, amino acids 1 to 930of SEQ ID NO: 75; a hexon protein of SAdV-26, amino acids 1 to 937 ofSEQ ID NO: 172; (b) a penton protein of SAdV-39, amino acids 1 to 532 ofSEQ ID NO: 6; a penton protein of SAdV-30, amino acids 1 to 533 of SEQID NO: 103; a penton protein of SAdV-25.2, amino acids 1 to 531 of SEQID NO: 135; a penton protein of SAdV-37, amino acids 1 to 542 of SEQ IDNO: 38; a penton protein of SAdV-38, amino acids 1 to 539 of SEQ ID NO:70; a penton protein of SAdV-26, amino acids 1 to 546 of SEQ ID NO: 167;and (c) a fiber protein of SAdV-39, amino acids 1 to 489 of SEQ ID NO:22, a fiber protein of SAdV-30, amino acids 1 to 445 of SEQ ID NO: 118;a fiber protein of SAdV-25.2, amino acids 1 to 443 of SEQ ID NO: 151; afiber protein of SAdV-37, amino acids 1 to 445 of SEQ ID NO: 54; a fiberprotein of SAdV-38, amino acids 1 to 425 of SEQ ID NO: 85; a fiberprotein of SAdV-26, amino acids 1 to 425 of SEQ ID NO: 183; and saidcapsid encapsidating a heterologous molecule carrying a gene operablylinked to expression control sequences which direct transcription,translation, and/or expression thereof in a host cell.
 2. The adenovirusaccording to claim 1, further comprising 5′ and 3′ adenoviruscis-elements necessary for replication and encapsidation.
 3. Theadenovirus according to claim 1, wherein said adenovirus lacks all or apart of the E1 gene.
 4. The adenovirus according to claim 3, whereinsaid adenovirus is replication-defective.
 5. The adenovirus according toclaim 5, wherein said virus has a hybrid capsid.
 6. The adenovirusaccording to claim 5, wherein said vector comprises more than one capsidprotein selected from SAdV-39, SAdV-30, SAdV-25.2, SAdV-37, SAdV-38, andSAdV-26.
 7. A recombinant adenovirus having a capsid comprising a hexoncontaining a fragment of a simian adenovirus hexon protein and a nucleicacid sequence heterologous to the SAdV, wherein the fragment of the SAdVhexon protein is the SAdV hexon protein of SEQ ID NO:11, 108, 140, 43,75 or 172 with an N-terminal or C-terminal truncation of about 50 aminoacids in length or is selected from the group consisting of: amino acidresidues 125 to 443 of SEQ ID NO:11, 108, 140, 43, 75 or 172; amino acidresidues 138 to 441 of SEQ ID NO:11, 108, 140, 43, 75 or 172; amino acidresidues 138 to 163 of SEQ ID NO:11, 108, 140, 43, 75 or 172; amino acidresidues 170 to 176 of SEQ ID NO:11, 108, 140, 43, 75 or 172; and aminoacid residues 404 to 430 of to SEQ ID NO: 11, 108, 140, 43, 75 or 172.8. The recombinant adenovirus according to claim 7, wherein the capsidfurther comprises a SAdV-39, SAdV-30, SAdV-25.2, SAdV-37, SAdV-38 orSAdV-26 fiber protein.
 9. The recombinant adenovirus according to claim7, wherein the capsid further comprises a SAdV-39, SAdV-30, SAdV-25.2,SAdV-37, SAdV-38 or SAdV-26 penton protein.
 10. The recombinantadenovirus according to claim 7, wherein said adenovirus is apseudotyped adenovirus comprising 5′ and 3′ adenovirus cis-elementsnecessary for replication and encapsidation, said cis-elementscomprising an adenovirus 5′ inverted terminal repeat and an adenovirus3′ inverted terminal repeat.
 11. The recombinant adenovirus according toclaim 7, wherein the adenovirus comprises a nucleic acid sequenceencoding a product operatively linked to sequences which directexpression of said product in a host cell.
 12. The recombinantadenovirus according to claim 7, wherein the recombinant adenoviruscomprises one or more adenovirus genes.
 13. The recombinant adenovirusaccording to claim 7, wherein the recombinant adenovirus isreplication-defective.
 14. The recombinant adenovirus according to claim13, wherein the recombinant adenovirus is deleted in adenovirus E1.15-16. (canceled)
 17. An isolated simian adenovirus nucleic acidselected from the group consisting of: simian adenovirus 39 nucleicacids 1 to 36553 of SEQ ID NO:1 and its complement; simian adenovirus25.2 nucleic acids 1 to 36629 of SEQ ID NO: 130 and its complement;simian adenovirus 26 nucleic acids 1 to 36628 of SEQ ID NO: 162 and itscomplement; simian adenovirus 30 nucleic acids 1 to 36621 of SEQ ID NO:98 and its complement; simian adenovirus 37 nucleic acids 1 to 36634 ofSEQ ID NO: 33 and its complement; simian adenovirus 38 nucleic acids 1to 36494 of SEQ ID NO: 65 and its complement.
 18. A vector comprising asimian adenovirus nucleic acid sequence selected from one or more of thegroup consisting of: (a) 5′ inverted terminal repeat (ITR) sequences;(b) the adenovirus E1 a region; (c) the adenovirus E1 region, or afragment thereof selected from among the group consisting of the openreading frames for the small T, large T, IX, and IVa2 regions; (d) theE2b region, including the open reading frames for the pTP; polymerase,and IVa regions; (e) the L1 region, or a fragment thereof selected fromamong the group consisting of the open reading frames for the 52/55 kDprotein, and IIIa protein; (f) the L2 region, or a fragment thereofselected from the group consisting of the open reading frames for thepenton, VII, VI, and X proteins; (g) the L3 region, or a fragmentthereof selected from the group consisting of the open reading framesfor the VI, hexon, or endoprotease; (h) the E2a protein, including theopen reading frame for the DNA-binding protein (DBP); (i) the L4 region,or a fragment thereof selected from the group consisting of the openreading frames for the 100 kD protein, the 33 kD homolog, the 22 kDhomolog, and VIII; (j) the E3 region, or a fragment thereof selectedfrom the group consisting of the open reading frames for the 12.5 K,CR1-alpha, gp19K, CR1-beta, CR1-gamma, CR1-delta, RID-alpha, RID-beta,and 14.7K; (k) the L5 region, or a fragment thereof selected from theopen reading frame for the fiber protein; (l) the E4 region, or afragment thereof selected from the group consisting of the open readingframes for the E4 ORF6/7, E4 ORF6, E4 ORF4, E4 ORF3, E4 ORF2, and E4ORF1; and (m) the 3′ ITR, of simian adenovirus 39, SEQ ID NO:1;SAdV-25.2, SEQ ID NO: 130; simian adenovirus 26, SEQ ID NO: 162, simianadenovirus 30, SEQ ID NO: 98; simian adenovirus 37, SEQ ID NO: 33;simian adenovirus 38, SEQ ID NO:
 65. 19. A simian adenovirus proteinencoded by the nucleic acid sequence according to claim
 18. 20. Acomposition comprising one or more simian adenovirus protein selectedfrom the group consisting of: E1a, selected from the amino acid sequenceof SEQ ID NO:30, 127, 159, 62, 95 and 191; E1, small T/19K, selectedfrom the amino acid sequence of SEQ ID NO:24, 120, 153, 56, 89, and 185;E1, large T/55K, selected from the amino acid sequence of SEQ ID NO: 2,99, 131, 34, 66, and 163; IX, selected from the amino acid sequence ofSEQ ID NO:3, 100, 132, 35, 67, and 164; 52/55D, selected from the aminoacid sequence of SEQ ID NO:4, 101, 133, 36, 68, and 165; IIIa, selectedfrom the amino acid sequence of SEQ ID NO:5, 102, 134, 37, 69 and 166;Penton, selected from the amino acid sequence of SEQ ID NO:6, 103, 135,38, 70, and 167; VII, selected from the amino acid sequence of SEQ IDNO: 7, 104, 136, 39, 71, and 168; V, selected from the amino acidsequence of SEQ ID NO: 8, 105, 137, 40, 72, and 169; pX, selected fromthe amino acid sequence of SEQ ID NO: 9, 106, 138, 41, 73, and 170; VI,selected from the amino acid sequence of SEQ ID NO: 10, 107, 139, 42,74, and 171; Hexon, selected from the amino acid sequence of SEQ ID NO:11, 108, 140, 43, 75, and 172; Endoprotease, selected from the aminoacid sequence of SEQ ID NO:12, 109, 141, 44, 76, and 173; 100 kD,selected from the amino acid sequence of SEQ ID NO:13, 110, 142, 45, 77,and 174; 33 kD, selected from the amino acid sequence of SEQ ID NO: 32,129, 161, 64, 97, 97, and 193; 22 kD, selected from the amino acidsequence of SEQ ID NO: 26, 122, 155, 58, 91, and 187; VIII, selectedfrom the amino acid sequence of SEQ ID NO:14, 111, 143, 46, 78, and 175;E3/12.5 K, selected from the amino acid sequence of SEQ ID NO:15, 123,144, 47, 79, and 176; CR1-alpha, selected from the amino acid sequenceof SEQ ID NO:27, 112, 156, 59, 92, and 188; gp19K, selected from theamino acid sequence of SEQ ID NO:16, 124, 145, 48, 87, and 177;CR1-beta, selected from the amino acid sequence of SEQ ID NO:17, 113,146, 49, 80, and 178; CR1-gamma, selected from the amino acid sequenceof SEQ ID NO:18, 114, 147, 50, 81, and 179; CR1-delta, selected from theamino acid sequence of SEQ ID NO:19, 115, 148, 51, 82, 180; RID-alpha,selected from the amino acid sequence of SEQ ID NO:20, 116, 149, 52, 83,and 181; RID-beta, selected from the amino acid sequence of SEQ IDNO:21, 117, 150, 53, 93, and 182; E3/14.7K, selected from the amino acidsequence of SEQ ID NO:28, 125, 158, 60, 84, and 189; and Fiber, selectedfrom the amino acid sequence of SEQ ID NO:22, 118, 151, 54, 85 and 183.21. A method for targeting a cell having an adenoviral receptorcomprising delivering to a subject a composition according to claim 20,said composition one or more simian adenovirus SAdV-39, -25.2, -26, -30,-37, and -38 proteins selected from a hexon, a penton and a fiber.
 22. Acomposition comprising a virus according to claim 1 in apharmaceutically acceptable carrier.
 23. A method for targeting a cellhaving an adenoviral receptor comprising delivering to a subject a virusaccording to claim 1.